Nanoscale platinum compounds and methods of use thereof

ABSTRACT

The invention is directed to biocompatible conjugated polymer nanoparticles including a copolymer backbone, a plurality of sidechains covalently linked to said backbone, and a plurality of platinum compounds dissociably linked to said backbone. The invention is also directed to dicarbonyl-lipid compounds wherein a platinum compound is dissociably linked to the dicarbonyl compound. The invention is also directed to methods of treating cancer or metastasis. The methods includes selecting a subject in need of treatment for cancer or metastasis and administering to the subject an effective amount of any of the nanoparticles, compounds, or compositions of the invention.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/147,751, filed on Mar. 16, 2012, which is a 35 U.S.C. §371National Stage Entry application of International Application No.PCT/US2010/023217, filed Feb. 4, 2010, which designates the U.S., andwhich claims benefit under 35 U.S.C. §119(e) of the U.S. ProvisionalApplication No. 61/149,725, filed Feb. 4, 2009 and 61/240,007, filedSep. 4, 2009, the content of all of which are incorporated herein byreference in their entirety.

GOVERNMENT SUPPORT

The subject matter of this application was made with support of aDepartment of Defense Era of Hope Scholar Award W81XWH-07-1-0482 andDepartment of Defense Postdoctoral Award W81XWH-09-1-0728. The U.S.Government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to biocompatible conjugated polymer nanoparticlesincluding a copolymer backbone, a plurality of sidechains covalentlylinked to the backbone, and, a plurality of platinum compoundsdissociably linked to the backbone.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of mortality in the United States,with an estimated 1,444,180 new cases and 565,650 deaths in 2008.Cytotoxic agents, which are used in standard chemotherapy,non-specifically target all dividing cells resulting in dose-limitingtoxicities. There is an urgent need to develop novel strategies that aremore specifically targeted against the tumor.

The use of nanovectors holds the potential to revolutionize cancerchemotherapy by specifically targeting the tumor. A number of polymericnanovectors are currently in development or in clinics, and aredramatically altering the pharmacodynamic and pharmacokinetic profile ofthe active agent. However, most of these polymeric constructs decreasethe potency of the conjugated active agent, relying on increased uptakeinto the tumor for the improved therapeutic index.

Cisplatin is one of the mainstays in chemotherapy regimes for most typesof Cancer (Kelland L. The resurgence of platinum-based cancerchemotherapy. Nat Rev Cancer. 2007 Aug. 7(8):573-84). However, its useis does-limited due to severe nephrotoxicity. Furthermore, thenanovector formulation of cisplatin, which is a first-line therapy formultiple cancers, has been a challenge.

SUMMARY OF THE INVENTION

Reported herein is a rational engineering of a polymeric construct ofplatinum-based chemotherapeutics such as cisplatin and oxaliplatin,which results in self-assembly into a nanoparticle. The nanoparticlesustains the potency of the active agent, and compared with cisplatin oroxaliplatin or carboplatin, exhibits increased anti-tumor effects withreduced systemic- and nephro-toxicity when administered intravenously totumor-bearing mice. This nanotechnology-enabled improvement in thetherapeutic index of cisplatin or oxaliplatin can be utilized for usingnanoplatinates in the clinical management of multiple types of cancer.

The invention is directed to a biocompatible conjugated polymernanoparticle including a copolymer backbone, a plurality of sidechainscovalently linked to said backbone, and, a plurality of platinumcompounds dissociably linked to said backbone. Generally, the platinumcompound is dissociably linked to backbone via linkage through thesidechain. In some embodiments, the platinum compound is linked to thesidechain through at least one coordination bond.

Another aspect of the invention is directed to biocompatible conjugatedpolymer nanoparticles including a polymaleic acid (PMA) such as apoly(isobutylene-alt-maleic acid) (PIMA) backbone. The backbone consistsof from 25 to 50 monomers. Also included are a plurality of PEGsidechains covalently linked to said backbone. The PEG sidechains have amolecular weight of from 200 to 3000 Dalton. The PEG sidechains numberbetween 50% and 100%, inclusive, of the number of monomeric units of thepolymer backbone. Also included are a plurality of cisplatin oroxaliplatin side groups dissociably linked to the backbone. Thecisplatin side groups number between 25% and 75%, inclusive, of thenumber of monomeric units of the polymer backbone.

Yet another aspect of the invention is directed to biocompatibleconjugated polymer nanoparticles including a poly(isobutylene-alt-maleicacid) backbone. The backbone consist of about 40 monomers. Also includedare a plurality of PEG sidechains covalently linked to the backbone. ThePEG sidechains have a molecular weight of about 2000 Dalton. The PEGsidechains number greater than 90% of monomeric units of said polymerbackbone. Also included are a plurality of cisplatin or oxaliplatin sidegroups dissociably linked to the backbone. The cisplatin or oxaliplatinside groups number between 25% and 75%, inclusive, of the number ofmonomeric units of the polymer backbone.

Still another aspect of the invention is directed to biocompatibleconjugated polymer nanoparticles including a poly(isobutylene-alt-maleicacid) backbone. The backbone consists of from 25 to 50 monomers. Alsoincluded are a plurality of glucosamine sidechains covalently linked tosaid backbone. The glucosamine sidechains number between 50% and 100%,inclusive, of monomeric units of said polymer backbone. Also includedare a plurality of cisplatin or oxaliplatin side groups dissociablylinked to the backbone. The cisplatin or oxaliplatin side groups numberbetween 25% and 75%, inclusive, of the number of monomeric units of thepolymer backbone.

Another aspect of the invention is directed to biocompatible conjugatedpolymer nanoparticles including a poly(isobutylene-alt-maleic acid)backbone. The backbone consists of from 25 to 50 monomers. Also includedare a plurality of glucosamine sidechains covalently linked to saidbackbone. The glucosamine sidechains number greater than 75% ofmonomeric units of said polymer backbone. Also included are a pluralityof cisplatin or oxaliplatin side groups dissociably linked to thebackbone. The cisplatin or oxaliplatin side groups number between 25%and 75%, inclusive, of the number of monomeric units of the polymerbackbone.

Yet another aspect of the invention is directed to carboxylicacid-platinum II (Pt(II)) complex conjugated nanoparticles including acarboxylic acid-(Pt(II)) complex and a plurality of lipid-polymerchains. The carboxylic acid portion of said carboxylicacid-cisplatin/oxaliplatin complex is covalently bound to saidlipid-polymer chains.

Another aspect of the invention is directed to a vesicle, micelle, orliposome compound comprising a plurality of nanoparticles of claim asdescribed herein.

Still another aspect of the invention is directed to pharmaceuticalcompositions including any of the nanoparticles or compounds describedherein and a pharmaceutically acceptable carrier.

Yet another aspect of the invention is directed to a method of treatingcancer or metastasis. The method includes selecting a subject in need oftreatment for cancer or metastasis and administering to the subject aneffective amount of any of the nanoparticles, compounds, or compositionsdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic of synthesis of PMA-Cisplatin. FIG. 1B showsthat loading of different numbers of cisplatin per polymer affects thesize of the nanoparticles as measured using DLS or TEM. FIG. 1C shows agraph of Cytotoxicity study of Polyisobutylene maleic acid carrier(PIMA) 2 and conjugate (PIMA-CISPLATIN) (6).

FIG. 2 shows a scheme showing derivatization of PMA with EDA. Thederivatized polymer was used to synthesize the cisplatin-complex. Thegraph shows the effect of different treatments on LLC viabilityfollowing 48 hours of incubation.

FIG. 3 shows a scheme of the synthesis of PMA-GA-Cisplatin. Complexationwith cisplatin was carried out over a 48 hour period. This resulted inthe formation of nanoparticles in the size range of around 100 nm asseen from the DLS measurements.

FIG. 4A is a graph showing the amount of active cisplatin that isreleased from the PMA-GA-Cisplatin nanoparticle when incubated with LLClysate. FIG. 4B is a concentration-effect graph showing the effect ofdifferent treatments on the viability of Lewis Lung Carcinoma cells whenincubated with the active agents for 48 hours. Cell viability wasmeasured using an MTS assay.

FIGS. 5A-5B are line graphs and FIGS. 5C-5F are bar graphs showingefficacy and toxicity profile of free and nanoparticle cisplatin inLewis Lung carcinoma model. Tumors were induced by injecting LLC cellsin c57/BL6 mouse. The effect of treatments on tumor volume (FIG. 5A) andbody weight (FIG. 5B) over the treatment period is shown. FIG. 5C is abar graph showing the blood counts between the various treatment groups.The animals were dosed thrice (shown by arrows on x-axis). Data shownare mean±SE, n=4-8. The effect of treatment on the organ weight ofkidneys (FIG. 5D) and spleen (FIG. 5E) as a marker for nephrotoxicityand hematological toxicity is also shown (n=4-6). The images on top ofeach graph show representative organs from each treatment group. FIG. 5Fshows the biodistribution of Pt in kidney and tumor as measured usingICP-spectroscopy 24 h hours following administration of free orcisplatin nanoparticles (8 mg/kg cisplatin dose)

FIG. 6 is a scheme showing synthesis of PMA-GA-Cisplatin (8).

FIG. 7 shows the effect of PMA-PEG-Cisplatin on Lewis Lung Carcinoma.Cell were incubated for 48 hours with the drugs or vehicles and thentested for viability using an MTS assay.

FIG. 8A-8B show the effect of different treatments on tumor growth andbody weight in vivo respectively. Tumors were induced by injecting LLCcells in c57/BL6 mouse.

FIG. 9 is a graph showing the amount of platination of the polymerquantified using a uv-vis spectroscopy method.

FIG. 10 is a scheme showing synthesis of lipid maleic acid-cipslatincomplex, which can form micelles in water.

FIGS. 11A and 11B are schematics showing SAR-inspired engineering of acisplatin nanoparticle. FIG. 11A shows the mechanism underlying theintracellular activation of cisplatin through aquation. The leavinggroups of cisplatin and analogues are replaced with OH prior toDNA-binding. FIG. 11B shows the chemical synthesis of PIMA-cisplatin andPIMA-glucosamine (PIMA-GA)-cisplatin complex. Transformation ofpolymaleic anhydride (n=40) (1) to polymaleic acid [PIMA] (2) enablescomplexation of [NH₂]2Pt[OH]₂ through dicarboxylato bond (6).Derivatization of one arm of PIMA with glucosamine (4), and complexationwith [NH₂]₂Pt[OH]₂ can lead to two isomers (8) and (10) depending on pH,characterized by unique Pt NMR signatures (FIG. 11B).

FIGS. 12A and 12B show characterization of cisPt-NP. Increasing thenumber of Pt on a PIMA (n=40) backbone increased the size ofnanoparticle formed. At an optimal Pt to polymer ratio, the inventorsobtained nanoparticles smaller than 150 nm, the size cut-off below whichenables preferential homing to tumors. FIG. 12A shows thatderivatization of all the monomeric units of PIMA with glucosamine andsubsequent complexation with Pt forms nanoparticles smaller than 150 nm.FIG. 12B shows the total platinum loaded per mg of polymer at thisratio.

FIGS. 13A-13F are line graphs showing in vitro characterization ofcisplatin nanoparticles. FIGS. 13A and 13B show the concentration-effectof different treatments on cellular viability as measured using MTSassay. X-axis shows the equivalent concentrations of cisplatin. Whereblank polymeric controls were used, dose of polymer used was equivalentto that used to deliver that specific dose of cisplatin in the complexedform. PIMA was also derivatized with ethylene diamine to generatePIMA-EDA, which offers a similar complexation environment to platinum asPIMA-GA. Unlike PIMA-GA, PIMA-EDA exerted inherent toxicity.PIMA-GA-Cisplatin[acidic] refers to the isomer formed under acidiccomplexation environment while PIMA-GA-Cisplatin[basic] refers to theisomer formed under alkaline environment. FIGS. 13C-13F show effect ofPIMA-GA-cisplatin nanoparticles on LLC cells viability, when the PIMAbackbone (40 monomeric units) is derivatized to different degrees.PIMA-30GA-Cisplatin has 30 of the 40 monomeric units derivatized withglucosamine while PIMA-GA-40 and PIMA-GA-200 have all the monomericunits derivatized. [a] and [b] refers to isomers formed in acidic andbasic environments when the polymers are complexed with cisplatin. Table1 shows the corresponding IC50 values.

FIGS. 14A-14H show FACS images and FIGS. 14I and 14J are bar graphsshowing that treatment with PIMA-GA-Cisplatin induces cell death.Representative FACS images of 4T1 (FIGS. 14A-14D) and LLC (FIGS.14E-14H) cells show the percentage in each quadrant following treatmentswith free or nanoparticle-cisplatin. Carboplatin was used as a controlfor comparison (FIGS. 14D and 14H). The cells were incubated with thedrugs for 24 h, following which they were labeled with Annexin-V FITCand counterstained with propidium iodide.

FIG. 15 is a schematic showing the labeling of the PIMA-GA polymer withFITC to enable the tracking of cellular uptake of the nanoparticles.

FIG. 16 is a line graph showing the effect of pH and Pt complexationenvironment on release kinetics. The nanoparticles were incubated at pH5.5 or pH8.5 in a dialysis bag, and release over time was quantified.The nanoparticles [PIMAGA-Cisplatin (O->Pt)] used were generated bycomplexing the polymer and cisplatin in an acidic pH [6.4] unless in thecase of PIMA-GA-Cisplatin (Pt->N), where the complexation was carriedout in a basic pH to generate the stable isomer [PIMA-GA-Cisplatin(N->Pt)]. The data shown are mean±SE from n=3.

FIGS. 17A-17B are line graphs and FIGS. 17C-17D are bar graphs showingPIMA-GA-cisplatin nanoparticle exerts similar anti-tumor effect withreduced systemic toxicity compared to free cisplatin in a 4T1 breastcancer model. Line graphs show the effect of treatments on tumor volume(FIG. 17A) and body weight (FIG. 17B) over the treatment period. Theanimals were dosed thrice (shown by arrows on x-axis). Data shown aremean±SE, n=4-8. Bar graphs show the effect of treatment on the organweight of spleen (FIG. 17C) and kidneys (FIG. 17D) as a marker fornephrotoxicity and hematological toxicity (n=4-6)*P<0.05 vsvehicle-treated group [ANOVA followed by Newman Keuls Post Hoc test].Carboplatin [3 mg/kg] was used as a control.

FIG. 18A is a bar graph and FIG. 18B is a line graph showingPIMA-GA-cisplatin nanoparticle inhibits tumor growth in aK-ras^(LSL/+)/Pten^(fl/fl) h ovarian cancer model. As shown in FIG. 18A,bioluminescence quantification indicated a significantly decreased tumorluciferase signal in mice treated with cisplatin-NP compared to vehicle(p<0.05, one-way ANOVA analysis). FIG. 18B shows drug toxicity assessedby measurements in overall body weight. Daily recording of body weightsindicated a significant loss of body weight in the free cisplatin groupas compared to both cisplatin-NP (1.25 mg/kg and 3 mg/kg) treated groups(P<0.05, two-way ANOVA analysis).

FIGS. 19A and 19B are bar graphs showing the distribution of Ptfollowing administration of cisplatin, cisplatin-nanoparticle([PIMA-GA-Cisplatin (O->Pt)] or carboplatin in breast cancer and ovariancancer respectively. Treatment was administered as described in FIGS.17A-D and 18A-B. The level of Pt in different tissues harvestedfollowing necropsy was quantified using inductively coupledplasma-spectrometry (ICP).

FIGS. 20A and 20B are schematic showing SAR-inspired engineering of aoxaliplatin nanoparticle. FIG. 20A shows the mechanism underlying theintracellular activation of oxaliplatin through aquation. FIG. 20B showsthe chemical synthesis of PIMA-oxaliplatin and PIMA-glucosamine(PIMA-GA)-oxaliplatin complex. Oxaliplatin-OH can be complexed with PIMAthrough dicarboxylato bonds. Derivatization of one arm of PIMA withglucosamine, and complexation with oxaliplatin can lead to two isomersdepending on pH.

FIGS. 21A and 21B are line graphs showing the concentration-effect ofdifferent treatments on cellular viability as measured using MTS assay.Breast cancer cell line, Lewis lung cancer (FIG. 21A) and 4T1 (FIG. 21B)cell lines were used for this study. X-axis shows the equivalentconcentrations of platinum. Where blank polymeric controls were used,dose of polymer used was equivalent to that used to deliver thatspecific dose of oxaliplatin in the complexed form. PIMA-GA-Ox refers tothe isomer [PIMA-GA-Oxaliplatin (O->Pt)] formed under acidiccomplexation environment. The PIMA-GA-oxaliplatin curve shifted to theleft, indicating that the nanoparticles are more effective than freeoxaliplatin in anti-tumor efficacy.

FIGS. 22A-22B are line graphs and FIGS. 22C-22E are bargraphs, showingPIMA-GA-oxaliplatin nanoparticle exerts similar anti-tumor effect withreduced systemic toxicity compared to free oxaliplatin in a 4T1 breastcancer model. Line graphs show the effect of treatments on tumor volume(FIG. 22A) and body weight (FIG. 22B) over the treatment period. Theanimals were dosed thrice. Data shown are mean±SE, n=4-8. Bar graphsshow the effect of treatment on the weight of tumor (FIG. 22C), kidney(FIG. 22D), and spleen (FIG. 22E) as a marker for nephrotoxicity andhematological toxicity (n=4-6).

FIG. 23 is a line graph showing concentration-effect of differentoxaliplatin complexes on cellular viability as measured using MTS assay.

FIG. 24 is a line graph showing the effect of cisplatin, carboplatin andPIMA-GA-200(A) on cell viability.

FIG. 25A is a scheme showing the synthesis of cholesterol-succinic acidconjugate and the complexation of Pt to the conjugate.

FIG. 25B shows a dynamic laser light scatter of liponanoparticles. Thesize of the liponanoparticles is less than 150 nm.

FIG. 26 is a line graph showing the release kinetics of Pt from theliponanoparticle with time and the influence of pH. The rate of releaseis faster in an acidic pH, which facilitates selective release of activeplatinate in the tumor, consistent with the acidic intratumoral pH.

FIGS. 27A-27C are line graphs showing the effect ofcisplatin-liponanoparticle on viability of 4T1 breast cancer cells. Cellviability was measured using MTS assay. The treatment withliponanoparticles results in rapid cell kill within 12 hours as comparedwith either cisplatin or carboplatin (FIG. 27A). At all three timepoints cisplatin-liponanoparticle was found to be more effective thancisplatin. Carboplatin is the least effective of all platinates tested(FIGS. 27A-27C).

FIGS. 28A and 28B are line graphs showing the effect ofcisplatin-liponanoparticle on viability of a cisplatin-resistanthepatocellular cancer cell line (CP20) and on a Lewis lung cancer cellline (LLC) respectively. Cisplatin acts on CP20 only at the highestconcentration, while the cells are susceptible to thecisplatin-liponanoparticle (FIG. 28A). Carboplatin has no effect at thisconcentration range (FIGS. 28A and 28B). Cisplatinliponanoparticleexerted superior anticancer effect than free cisplatin on LLCs (FIG.28B).

FIGS. 29A-29B are line graphs and FIGS. 29C-29E are bar graphs showingthe efficacy of cisplatin-liponanoparticle in a 4T1 syngeneic tumormodel in vivo. The graphs show the effect of different treatments ontumor growth (FIGS. 29A and 29C) and body weight (as a marker forsystemic toxicity, FIG. 29B). Bar graphs show kidney (FIG. 29D) andspleen (FIG. 29E) weights as markers for nephrotoxicity andhematological toxicity. As seen in the FIGS. 29A-29E,cisplatin-liponanoparticle induced greater antitumor activity withreduced systemic, nephrotoxicity.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to biocompatible conjugated polymernanoparticles including a copolymer backbone, a plurality of sidechainscovalently linked to said backbone, and, a plurality of platinumcompounds dissociably linked to said backbone. Generally, the platinumcompounds are linked to the backbone via a linkage to the sidechains.

In some embodiments, the copolymer comprises maleic acid monomers.

In a preferred embodiment, the copolymer is poly(isobutylene-alt-maleicacid) (PIMA or PMA).

In some embodiments, the copolymer comprises from 2 to 100 monomericunits. In some embodiments, the copolymer comprises from 25 to 50monomeric units.

In some embodiments, the sidechains are selected from the groupconsisting of polymers, monosaccharides, carboxylic acids, dicarboxylicacids, amides, and combinations thereof.

In preferred embodiments, the sidechains are polyethylene glycol (PEG).PEG sidechains can be represented by —C(O)—NH-PEG.

In some embodiments, the PEG sidechains have a molecular weight of from100 to 5000 Dalton. In some embodiments, the PEG sidechains have amolecular weight of from 1000 to 3000 Dalton. In a preferred embodiment,the PEG sidechains have a molecular weight of about 2000 Dalton.

In some embodiments, the sidechains are monosaccharides. In a preferredembodiment, the monosaccharides are glucosamine. The monosaccharidesidechains can be represented by —C(O)-saccharide.

Any platinum compound can be used in the invention. Preferably, theplatinum compound is a platinum(II) or platinum (IV) compound. In someembodiments, the platinum (II) compound is selected from the groupconsisting of cisplatin, oxaliplatin, carboplatin, paraplatin,sartraplatin, and combinations thereof. In a preferred embodiment, theplatinum (II) compound side group is cisplatin or oxalipaltin.

In some embodiments, the platinum(II) compound is selected from thegroup consisting of Pt(NH₃)₂, Pt(NH₃)(2-methylpyridine), and

wherein p is 0-3. In a preferred embodiment, the platinum (II) compoundis Pt(NH₃)₂.

In some embodiments, the platinum (II) compound is

wherein p is 0-3.

In some embodiments, the platinum (II) compound comprises at least twonitrogen atoms, where said nitrogen atoms are directly linked toplatinum. In a further embodiment, the two nitrogen atoms are linked toeach other via an optionally substituted linker, e.g. acyclic or cycliclinker. A cyclic linker means a linking moiety that comprises at leastone ring structure. Cyclic linkers can be aryl, heteroaryl, cyclyl orheterocyclyl.

In some embodiments, at least one nitrogen that is linked to platinum isa ring atom of a heteroaryl or a heterocyclyl. In a preferredembodiment, heteroaryl is optionally substituted pyridine, e.g.,2-methylpyridine.

In some embodiments, the plurality of sidechains corresponds to a numberbetween 50% and 100%, inclusive, of the number of monomeric units ofsaid polymer backbone. This means that between 50% to 100% of themonomeric units have at least one sidechain linked to the monomericunit. The total number of sidechains can be greater than the totalnumber of the monomeric units. For example, two sidechains can beattached to a maleic acid monomer.

In some embodiments, the plurality of sidechains corresponds to a numbergreater than 90% of the number of monomeric units of said polymerbackbone.

In some embodiments, the plurality of platinum compounds corresponds tonumber between 10% and 100%, inclusive, of the number of monomeric unitsof said polymer backbone. Generally there is a one-to-one relationshipbetween platinum compounds and monomeric subunits. Thus, the percentrefers to the number of monomeric units that are linked to a platinumcompound to the total number of monomeric units present in the polymerbackbone.

In some embodiments, the plurality of platinum compounds corresponds tonumber between 25% and 75%, inclusive, of the number of monomeric unitsof said polymer backbone.

Generally from 10 to 500 ug of the platinum compound can be loaded on 1mg of the polymer. Preferably, 50 to 250 ug, more preferably 150 to 200ug of the platinum compound is loaded on 1 mg of the polymer. In someexperiments, the inventors obtained a loading of 175±5 ug/mg of polymer.

In some embodiments, the sidechains comprise dicarboxylic acids. In someembodiments, the dicarboxylic acids are of the formula HOOC—R—COOH,wherein R is a C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl. In apreferred embodiment, the dicarboxylic acid is maleic acid.

In some embodiments, the copolymer comprises at least one monomer havingthe formula —CH(CO₂H)—R—CH(C(O)R′)—, wherein R is a bond, C₁-C₆alkylene, where the alkylene can comprise one or more double or triplebonds; and R′ is a substituted nitrogen atom. Preferably, R is a bond.

In some embodiments, between 50% to 100%, inclusive, of the monomericsubunits in the polymeric backbone are —CH(CO₂H)—R—CH(C(O)R′)—, whereinR is a bond, C₁-C₆ alkylene, where the alkylene can comprise one or moredouble or triple bonds; and R′ is a substituted nitrogen atom.

In some embodiments, at least 90% or more of the monomeric subunits inthe polymeric backbone are —CH(CO₂H)—R—CH(C(O)R′)—, wherein R is a bond,C₁-C₆ alkylene, where the alkylene can comprise one or more double ortriple bonds; and R′ is a substituted nitrogen atom.

In some embodiments, the copolymer comprises at least one monomer havingthe formula —CH(CO₂H)—R—CH(C(O)R′)CH₂C(Me₂)- or—CH(C(O)R′)—R—CH(CO₂H)—CH₂C(Me₂)-, wherein R is a bond, C₁-C₆ alkylene,where the alkylene can comprise one or more double or triple bonds; andR′ is a substituted nitrogen atom. Preferably, R is a bond.

In some embodiments, the copolymer comprises between 50% to 100%,inclusive of monomers having the formula—CH(CO₂H)—R—CH(C(O)R′)CH₂C(Me₂)- or —CH(C(O)R′)—R—CH(CO₂H)—CH₂C(Me₂)-,wherein R is a bond, C₁-C₆ alkylene, where the alkylene can comprise oneor more double or triple bonds; and R′ is a substituted nitrogen atom.

In some embodiments, the copolymer comprises at least 90% of monomershaving the formula —CH(CO₂H)—R—CH(C(O)R′)CH₂C(Me₂)- or—CH(C(O)R′)—R—CH(CO₂H)—CH₂C(Me₂)-, wherein R is a bond, C₁-C₆ alkylene,where the alkylene can comprise one or more double or triple bonds; andR′ is a substituted nitrogen atom.

In some embodiments, R′ is

or —NH(CH₂CH₂O)_(m)CH₃, wherein m is 1-150.

In some embodiments, at least one monomer of the polymer comprises twosidechains selected from the group consisting of carboxylic acid, amide,and ester. Said sidechains being separated from each other by 1, 2, 3,4, 5, 6, 7, 8, 9 or ten carbon, oxygen, nitrogen, sulfur atoms, orcombination thereof. Preferably, said amide and ester sidechains areseparated from each other by two carbon atoms. Preferably, at least oneof the sidechains is not a carboxylic acid.

In some embodiments, at least one monomer of the polymer comprises twocarboxylic acid sidechains. Said carboxylic acid sidechains beingseparated from each other by 1, 2, 3, 4, 5, 6, 7, 8, 9 or ten carbon,oxygen, nitrogen, sulfur atoms, or combination thereof. Preferably, saidcarboxylic acid sidechains are separated from each other by two carbonatoms. The said carbon atoms can be linked to each other through asingle bond or a double bond.

In some embodiments, at least one monomer of the polymer comprises acarboxylic acid and an amide sidechains. Said carboxylic acid sidechainsand amide sidechains being separated from each other by 1, 2, 3, 4, 5,6, 7, 8, 9 or ten carbon, oxygen, nitrogen, sulfur atoms, or combinationthereof. Preferably, said carboxylic acid and amide sidechains areseparated from each other by two carbon atoms. The said carbon atoms canbe linked to each other through a single bond or a double bond.

In some embodiments, at least one monomer of the polymer comprises acarboxylic acid and ester sidechains. Said carboxylic acid sidechainsand ester sidechains being separated from each other by 1, 2, 3, 4, 5,6, 7, 8, 9 or ten carbon, oxygen, nitrogen, sulfur atoms, or combinationthereof. Preferably, said carboxylic acid and ester sidechains areseparated from each other by two carbon atoms. The said carbon atoms canbe linked to each other through a single bond or a double bond.

In some embodiments, at least one monomer of the polymer comprises anamide and ester sidechains. Said amide sidechains and ester sidechainsbeing separated from each other by 1, 2, 3, 4, 5, 6, 7, 8, 9 or tencarbon, oxygen, nitrogen, sulfur atoms, or combination thereof.Preferably, said amide and ester sidechains are separated from eachother by two carbon atoms. The said carbon atoms can be linked to eachother through a single bond or a double bond.

In some embodiments, at least one monomer of the polymer comprises twoamide sidechains. Said amide sidechains being separated from each otherby 1, 2, 3, 4, 5, 6, 7, 8, 9 or ten carbon, oxygen, nitrogen, sulfuratoms, or combination thereof. Preferably, said amide and estersidechains are separated from each other by two carbon atoms.

In some embodiments, at least one monomer of the polymer comprises twoester sidechains. Said ester sidechains being separated from each otherby 1, 2, 3, 4, 5, 6, 7, 8, 9 or ten carbon, oxygen, nitrogen, sulfuratoms, or combination thereof. Preferably, said amide and estersidechains are separated from each other by two carbon atoms.

In some embodiments, the polymer comprises two sidechains selected fromthe group consisting of carboxylic acid, amide, and ester. Saidsidechains being separated from each other by 1, 2, 3, 4, 5, 6, 7, 8, 9or ten carbon, oxygen, nitrogen, sulfur atoms, or combination thereof.Preferably, said amide and ester sidechains are separated from eachother by two carbon atoms. Preferably, at least least one of thesidechains is not a carboxylic acid.

In some embodiments, the polymer comprises at least two carboxylic acidsidechains. Said carboxylic acid sidechains being separated from eachother by 1, 2, 3, 4, 5, 6, 7, 8, 9 or ten carbon, oxygen, nitrogen,sulfur atoms, or combination thereof. Preferably, said carboxylic acidsidechains are separated from each other by two carbon atoms.

In some embodiments, the polymer comprises a carboxylic acid and anamide sidechains. Said carboxylic acid sidechains and amide sidechainsbeing separated from each other by 1, 2, 3, 4, 5, 6, 7, 8, 9 or tencarbon, oxygen, nitrogen, sulfur atoms, or combination thereof.Preferably, said carboxylic acid and amide sidechains are separated fromeach other by two carbon atoms.

In some embodiments, the polymer comprises a carboxylic acid and anester sidechains. Said carboxylic acid sidechains and ester sidechainsbeing separated from each other by 1, 2, 3, 4, 5, 6, 7, 8, 9 or tencarbon, oxygen, nitrogen, sulfur atoms, or combination thereof.Preferably, said carboxylic acid and ester sidechains are separated fromeach other by two carbon atoms.

In some embodiments, the polymer comprises an amide and an estersidechains. Said amide sidechains and ester sidechains being separatedfrom each other by 1, 2, 3, 4, 5, 6, 7, 8, 9 or ten carbon, oxygen,nitrogen, sulfur atoms, or combination thereof. Preferably, said amideand ester sidechains are separated from each other by two carbon atoms.The said carbon atoms can be linked to each other through a single bondor a double bond.

In some embodiments, the polymer comprises two amide sidechains. Saidamide sidechains being separated from each other by 1, 2, 3, 4, 5, 6, 7,8, 9 or ten carbon, oxygen, nitrogen, sulfur atoms, or combinationthereof. Preferably, said amide and ester sidechains are separated fromeach other by two carbon atoms.

In some embodiments, the polymer comprises two ester sidechains. Saidester sidechains being separated from each other by 1, 2, 3, 4, 5, 6, 7,8, 9 or ten carbon, oxygen, nitrogen, sulfur atoms, or combinationthereof. Preferably, said amide and ester sidechains are separated fromeach other by two carbon atoms.

The nanoparticles of the invention can range in size from 25-250 nm,preferably 50-200 nm, more preferably 80-160 nm, and most preferably90-110 nm. Without wishing to be bound by theory, nanoparticles in thesize range of 80-160 home preferentially into tumors resulting from theenhanced permeability and retention. See for example, Moghimi, et al.,Pharmacol Rev. 2001 June; 53(2):283-318.

In some embodiments, the platinum compound is dissociably linked to saidbackbone through at least one coordination bond. Without wishing to bebound by theory, the coordination bond is more liable and thus releasesthe platinum compound more easily.

In some embodiments, linking of the platinum compound to the biopolymerbackbone further comprises a carboxylato bond. In some embodiments, theplatinum compound is linked to the backbone through a coordination bondand a carboxylato bond.

It is to be understood that although linkage to backbone is recited, oneof skill in the art understands that the platinum compound is generallylinked to one or more sidechains which themselves are linked to thebackbone. So any recitation of linking of a platinum compound tobackbone encompasses the situations where the platinum compound islinked to a sidechain which is then linked to the backbone.

In some embodiments, the coordination bond is between platinum atom ofthe platinum compound and an oxygen of the sidechain. Preferably thecoordination bond is between platinum and a carbonyl oxygen.

In some embodiments, the coordination bond is between platinum atom ofthe platinum compound and an amide oxygen of the sidechain. In someembodiments, the coordination bond is between platinum atom of theplatinum compound and an ester carbonyl oxygen of the sidechain.

In some embodiments, the copolymer comprises at least one maleic acidmonomer, wherein at least one carboxylic acid of said at least maleicacid is derivatized to an amide.

In some embodiments, between 50% to 100%, inclusive, of the monomericunits in the polymer backbone are maleic acid monomer and wherein atleast one carboxylic acid of said maleic acid monomer is derivatized toan amide.

In some embodiments, at least 90% of the monomeric units in the polymerbackbone are maleic acid monomer and wherein at least one carboxylicacid of said maleic acid monomer is derivatized to an amide.

The loading of platinum compound onto the polymer can be represented byas percent mg of platinum compound per mg of polymer. For example, amaximum of 0.375 mg of cisplatin can be loaded onto the PIMA-GA polymertherefore a loading of 37.5% represents the maximum loading for thatparticular polymer. The loading can range from about 1% to thetheoretical total loading for a polymer.

In some embodiments, the platinum compound loading is from 1%-37.5%. Thepercent loading represent mg of platinum compound linked with per mg ofpolymer.

In some embodiments, the platinum compound loading is from 1%-6%. Insome embodiments, the Pt(II) compound loading is from 0.01% to 1° 0%.

Another aspect of the invention is directed to biocompatible conjugatedpolymer nanoparticles including a poly(isobutylene-alt-maleic acid)backbone. The backbone consists of from 25 to 50 monomers. Also includedare a plurality of PEG sidechains covalently linked to said backbone.The PEG sidechains have a molecular weight of from 1000 to 3000 Dalton.The PEG sidechains number between 50% and 100%, inclusive, of the numberof monomeric units of the polymer backbone. Also included are aplurality of cisplatin side groups dissociably linked to the backbone.The cisplatin side groups number between 25% and 75%, inclusive, of thenumber of monomeric units of the polymer backbone.

Yet another aspect of the invention is directed to biocompatibleconjugated polymer nanoparticles including a poly(isobutylene-alt-maleicacid) backbone. The backbone consist of 40 monomers. Also included are aplurality of PEG sidechains covalently linked to the backbone. The PEGsidechains have a molecular weight of 2000 Dalton. The PEG sidechainsnumber greater than 90% of monomeric units of said polymer backbone.Also included are a plurality of cisplatin side groups dissociablylinked to the backbone. The cisplatin side groups number between 25% and75%, inclusive, of the number of monomeric units of the polymerbackbone.

Still another aspect of the invention is directed to biocompatibleconjugated polymer nanoparticles including a poly(isobutylene-alt-maleicacid) backbone. The backbone consists of from 25 to 50 monomers. Alsoincluded are a plurality of glucosamine sidechains covalently linked tosaid backbone. The glucosamine sidechains number between 50% and 100%,inclusive, of monomeric units of said polymer backbone. Also includedare a plurality of cisplatin side groups dissociably linked to thebackbone. The cisplatin side groups number between 25% and 75%,inclusive, of the number of monomeric units of the polymer backbone.

Another aspect of the invention is directed to biocompatible conjugatedpolymer nanoparticles including a poly(isobutylene-alt-maleic acid)backbone. The backbone consists of from 25 to 50 monomers. Also includedare a plurality of glucosamine sidechains covalently linked to saidbackbone. The glucosamine sidechains number greater than 90% ofmonomeric units of said polymer backbone. Also included are a pluralityof cisplatin side groups dissociably linked to the backbone. Thecisplatin side groups number between 25% and 75%, inclusive, of thenumber of monomeric units of the polymer backbone.

Yet another aspect of the invention is directed to carboxylicacid-platinum compound complex conjugated nanoparticles including acarboxylic acid-platinum compound complex and a plurality oflipid-polymer chains. The carboxylic acid portion of said carboxylicacid-platinum complex is covalently bound to said lipid-polymer chains.

In a preferred embodiment, the carboxylic acid is maleic acid. In someembodiments, the polymer is PEG.

In certain embodiments, the platinum compound loading is from 1%-37.5%.In certain embodiments, the platinum compound loading is from 1%-6%.

The platinum compound can be Pt(II) compound or a Pt(IV) compound. Insome embodiments, the Pt(II) compound is selected from the groupconsisting of cisplatin, oxaliplatin, carboplatin, paraplatin,sartraplatin, and combinations thereof. In a preferred embodiment, thePt(II) compound is cisplatin.

Another aspect of the invention is directed to a vesicle, micelle, orliposome compound comprising a plurality of nanoparticles of claim asdescribed herein.

Still another aspect of the invention is directed to a pharmaceuticalcomposition including any of the nanoparticles or compounds describedherein and a pharmaceutically acceptable carrier.

Yet another aspect of the invention is directed to a method of treatingcancer or metastasis. The method includes selecting a subject in need oftreatment for cancer or metastasis and administering to the subject aneffective amount of any of the nanoparticles, compounds, or compositionsdescribed herein.

In some embodiments, the cancer or metastasis is selected from the groupconsisting of platinum susceptible or resistant tumors including breast,head and neck, ovarian, testicular, pancreatic, oral-esophageal,gastrointestinal, liver, gall bladder, lung, melanoma, skin cancer,sarcomas, blood cancers, brain tumors including glioblastomas, andtumors of neuroectodermal origin.

In yet another aspect, the invention provide for methods of formulatingplatinum compound polymer nanoparticles, method comprising conjugationof platinum compound with a biocompatible polymer or biocompatiblecopolymer. Without wishing to be bound by theory, conjugation ofplatinum compound with the biocompatible polymer at acidic pH results innanoparticles that are more active in vivo than when conjugation is doneat basic pH.

Accordingly, in some embodiments, conjugation is done at pH below 7,preferably a pH between 1 and 6.9. In some more preferred embodiments,conjugation is carried out at a pH of 6.5.

The inventors have observed that conjugation under basic conditionsfavor the formation of an isomeric PIMA-GA_Cisplantin complex with amonocarboxylato and a more stable Pt<->N coordinate bond. In contrast,complexation PIMA-GA and cisplatin in an acidic pH generates theisomeric state characterized by the monocarboxylato bond and Pt<->Ocoordinate bond. Thus, conjugations conditions that lead to theformation of a Pt<->O coordinate bond over a Pt<->N coordinate bond arepreferred for conjugation.

Generally an excess of the Pt(II) compound to the polymer is used. Insome embodiments, 5-25 mole access of Pt(II) compound to polymer isused. Preferably 10-20 mole access of platinum(II) compound to polymeris used. In one preferred 15 mole access of Pt(II) compound to polymeris used.

In yet another aspect, the invention provides a dicarbonyl moleculelinked to a lipid molecule. Such a compound can be represented by thestructure lipid-linker-dicarbnoyl. These molecules can be used tocomplex platinum compounds such as cisplatin, oxaliplatin or otherplatinates and platinum compounds described herein throughcarboxylato-linkage and/or coordination bonds. These can then be mixedwith appropriate lipids/phospholipids to nanoparticles of less than 150nm, which release Pt in a pH-dependent manner. Once formulated thesenanoparticles exhibit improved efficacy and toxicity profile as comparedwith carboplatin and cisplatin, and are active in a cisplatin-resistantcancer.

These nanoparticles can be formulated to comprise pharmaceuticallyactive agents for delivery.

The term “Lipid” is used in the conventional sense to refer to moleculesthat are soluble to a greater or lesser degree in organic solvents, likealcohols, and relatively insoluble in aqueous media. Thus, the term“lipid” includes compounds of varying chain length, from as short asabout 2 carbon atoms to as long as about 28 carbon atoms. Additionally,the compounds may be saturated or unsaturated. and in the form ofstraight- or branched-chains or in the form of unfused or fused ringstructures. Exemplary lipids include, but are not limited to, fats,waxes, sterols, steroids, bile acids, fat-soluble vitamins (such as A,D, E, and K), monoglycerides, diglycerides, phospholipids, glycolipids,sulpholipids, aminolipids, chromolipids (lipochromes),glycerophospholipids, sphingolipids, prenol lipids, saccharolipids,polyketides, and fatty acids. In some embodiments, the lipid ischolesterol or distearoylphosphatidylethanolamine.

Generally any molecule that has two carbonyl groups can be used. In someembodiments, the dicarbonyl molecule is a dicarboxylic acid, or aketo-carboxylic acid. In some preferred embodiments, the dicarbonylmolecule is succinic acid.

In some embodiments, the dicarbonyl molecule is R′OC(O)—R—C(O)—, whereinR is C₁-C₆ alkylene, where the alkylene can comprise one or more doubleor triple bonds and/or the backbone of the alkylene can be interruptedwith one or more of O, S, S(O), SO₂, NH, C(O); and R′ is H, alkyl,alkenyl, alkynyl, aryl, heteroaryl, acylcy, heterocyclyl, each of whichcan be optionally substituted. Preferably R is CH₂, —CH₂CH₂—,—CH₂CH₂—CH₂— or —CH═CH—. Preferably R′ is H.

The dicarbonyl molecule can be linked with the lipid molecule directlyor through a linker molecule. The term “linker” means an organic moietythat connects two parts of a compound. Linkers typically comprise adirect bond or an atom such as oxygen or sulfur, a unit such as NH,C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,al kenylheteroarylalkenyl, alkenylheteroarylal kynyl,alkynylheteroarylal kyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, where one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, NH, C(O). It is to be understood that thediacarbonyl molecule and/or the lipid can be modified to comprisefunctional groups for linking to each other or to the linker.

In some embodiments, linker is a diamine such as ethylene diamine. Insome embodiments, linker is PEG-NH₂.

In one preferred embodiment, linker is —NHCH₂CH₂C(O)—. In anotherpreferred embodiment, linker is —CH₂CH₂NHC(O)—[OCH₂CH₂]_(z)—NH—, where zis 1-50. Preferably z is 45.

In some embodiments, the lipid-dicarbonyl compound is as shown in FIGS.10 (compound 2) and 25A (compound 5).

In another aspect, the invention provide a biocompatible polymercomprising at least one monomer having the formula—CH(CO₂H)—R—CH(C(O)R′)—, wherein R is a bond, C₁-C₆ alkylene, where thealkylene can comprise one or more double or triple bonds; and R′ is asubstituted nitrogen atom. Preferably, R is a bond.

In some embodiments, the polymer comprises from 2 to 100 monomeric unitshaving the formula —CH(CO₂H)—R—CH(C(O)R′)—, wherein R is a bond, C₁-C₆alkylene, where the alkylene can comprise one or more double or triplebonds; and R′ is a substituted nitrogen atom.

In some embodiments, the polymer comprises from 25 to 50 monomeric unitshaving the formula —CH(CO₂H)—R—CH(C(O)R′)—, wherein R is a bond, C₁-C₆alkylene, where the alkylene can comprise one or more double or triplebonds; and R′ is a substituted nitrogen atom.

In some embodiments, between 500/% to 100%, inclusive, of the monomericsubunits in the polymeric backbone are —CH(CO₂H)—R—CH(C(O)R′)—, whereinR is a bond, C₁-C₆ alkylene, where the alkylene can comprise one or moredouble or triple bonds; and R′ is a substituted nitrogen atom.

In some embodiments, at least 90% or more of the monomeric subunits inthe polymeric backbone are —CH(CO₂H)—R—CH(C(O)R′)—, wherein R is a bond,C₁-C₆ alkylene, where the alkylene can comprise one or more double ortriple bonds; and R′ is a substituted nitrogen atom.

In some embodiments, the copolymer comprises at least one monomer havingthe formula —CH(CO₂H)—R—CH(C(O)R′)CH₂C(Me₂)- or—CH(C(O)R′)—R—CH(CO₂H)—CH₂C(Me₂)-, wherein R is a bond, C₁-C₆ alkylene,where the alkylene can comprise one or more double or triple bonds; andR′ is a substituted nitrogen atom. Preferably, R is a bond.

In some embodiments, the copolymer comprises between 50% to 100%,inclusive of monomers having the formula—CH(CO₂H)—R—CH(C(O)R′)CH₂C(Me₂)- or —CH(C(O)R′)—R—CH(CO₂H)—CH₂C(Me₂)-,wherein R is a bond, C₁-C₆ alkylene, where the alkylene can comprise oneor more double or triple bonds; and R′ is a substituted nitrogen atom.

In some embodiments, the copolymer comprises at least 90% of monomershaving the formula —CH(CO₂H)—R—CH(C(O)R′)CH₂C(Me₂)- or—CH(C(O)R′)—R—CH(CO₂H)—CH₂C(Me₂)-, wherein R is a bond, C₁-C₆ alkylene,where the alkylene can comprise one or more double or triple bonds; andR′ is a substituted nitrogen atom.

In some embodiments, R′ is

or —NH(CH₂CH₂O)_(m)CH₃, wherein m is 1-150.

These polymers can be used for formulating nanoparticles and gels whichcan be used for drug delivery. Thus, the invention also providesnanoparticles comprising a polymer described herein and one or morebioactive active agent (“bioactive agent”).

Compositions described herein can be used in methods for sustainedrelease of bioactive active agents. In one embodiment, the methodcomprising: (a) providing or administering to a subject a compositiondescribed herein, wherein the composition contains the bioactive agent.As used herein, “bioactive agents” refer to naturally occurringbiological materials, for example, extracellular matrix materials suchas fibronectin, vitronection, and laminin; cytokins; and growth factorsand differentiation factors. “Bioactive agents” also refer toartificially synthesized materials, molecules or compounds that have abiological effect on a biological cell, tissue or organ.

Suitable growth factors and cytokines include, but are not limited, tostem cell factor (SCF), granulocyte-colony stimulating factor (G-CSF),granulocyte-macrophage stimulating factor (GM-CSF), stromal cell-derivedfactor-1, steel factor, VEGF, TGFβ, platelet derived growth factor(PDGF), angiopoeitins (Ang), epidermal growth factor (EGF), bFGF, HNF,NGF, bone morphogenic protein (BMP), fibroblast growth factor (FGF),hepatocye growth factor, insulin-like growth factor (IGF-1), interleukin(IL)-3, IL-1α, IL-1β, IL-6, IL-7, IL-8, IL-11, and IL-13,colony-stimulating factors, thrombopoietin, erythropoietin, fit3-ligand,and tumor necrosis factor α (TNFα). Other examples are described inDijke et al., “Growth Factors for Wound Healing”, Bio/Technology,7:793-798 (1989); Mulder G D, Haberer P A, Jeter K F, eds. Clinicians'Pocket Guide to Chronic Wound Repair. 4th ed. Springhouse, Pa.:Springhouse Corporation; 1998:85; Ziegler T. R., Pierce, G. F., andHerndon, D. N., 1997, International Symposium on Growth Factors andWound Healing: Basic Science & Potential Clinical Applications (Boston,1995, Serono Symposia USA), Publisher: Springer Verlag.

In some embodiments, suitable bioactive agents include but not limitedto therapeutic agents. As used herein, the term “therapeutic agent”refers to a substance used in the diagnosis, treatment, or prevention ofa disease. Any therapeutic agent known to those of ordinary skill in theart to be of benefit in the diagnosis, treatment or prevention of adisease is contemplated as a therapeutic agent in the context of thepresent invention. Therapeutic agents include pharmaceutically activecompounds, hormones, growth factors, enzymes, DNA, plasmid DNA, RNA,siRNA, viruses, proteins, lipids, pro-inflammatory molecules,antibodies, antibiotics, anti-inflammatory agents, anti-sensenucleotides and transforming nucleic acids or combinations thereof. Anyof the therapeutic agents may be combined to the extent such combinationis biologically compatible.

Exemplary therapeutic agents include, but are not limited to, thosefound in Harrison's Principles of Internal Medicine, 13^(th) Edition,Eds. T. R. Harrison et al. McGraw-Hill N.Y., NY; Physicians DeskReference, 50^(th) Edition, 1997, Oradell New Jersey, Medical EconomicsCo.; Pharmacological Basis of Therapeutics, 8^(th) Edition, Goodman andGilman, 1990; United States Pharmacopeia, The National Formulary, USPXII NF XVII, 1990; current edition of Goodman and Oilman's ThePharmacological Basis of Therapeutics; and current edition of The MerckIndex, the complete contents of all of which are incorporated herein byreference.

Examples of therapeutic agents which may be incorporated in thecomposition, include but are not limited to, narcotic analgesic drugs;salts of gold; corticosteroids; hormones; antimalarial drugs; indolederivatives; pharmaceuticals for arthritis treatment; antibiotics,including Tetracyclines, Penicillin, Streptomycin and Aureomycin;antihelmintic and canine distemper drugs, applied to domestic animalsand large cattle, such, as, for example, phenothiazine; drugs based onsulfur, such, as sulfioxazole; antitumor drugs; pharmaceuticalssupervising addictions, such as agents controlling alcohol addiction andagents controlling tobacco addiction; antagonists of drug addiction,such, as methadone; weightcontrolling drugs; thyroid gland controllingdrugs; analgesics; drugs controlling fertilization or contraceptionhormones; amphetamines; antihypertensive drugs; antiinflammatoriesagents; antitussives; sedatives; neuromuscular relaxants; antiepilepticdrugs; antidepressants; antidisrhythmic drugs; vasodilating drugs;antihypertensive diuretics; antidiabetic agents; anticoagulants;antituberculous agents; antipsyhotic agents; hormones and peptides. Itis understood that above list is not full and simply represents the widediversification of therapeutic agents that may be included in thecompositions. In some embodiments, therapeutic agent is Mitoxantrone,protein (e.g. VEGF) or plasmid DNA.

The amount of therapeutic agent distributed in a composition depends onvarious factors including, for example, specific agent; function whichit should carry out; required period of time for release of a the agent;quantity to be administered. Generally, dosage of a therapeutic agenti.e. amount of therapeutic agent in composition, is selected from therange about from 0.001% (w/w) up to 95% (w/w), preferably, from about 5%(w/w) to about 75% (w/w), and, most preferably, from about 10% (w/w) toabout 60% (w/w).

Cisplatin [cis-dichlorodiammineplatinum(II)] (CDDP) has emerged as animportant class of antitumor agents, and is widely used for thetreatment of many malignancies including testicular, ovarian, cervical,head and neck, and non-small cell lung cancer (Jamieson, et al, Chem.Rev. (1999), 99(9): 2467-2498). It was also shown to be active in triplenegative breast cancer (Leong, et al., J. Clin. Invest. (2007), 117(5):1370-80). Its use is however dose-limited mainly because ofnephrotoxicity or toxicity to the kidney (Madias, N E and Harrington, JT, Am. J. (1978), 65(2): 307-14). To address this limitation, twodirections of research has evolved, the first focused on the synthesisof platinum analogues, the second is to engineer novel nanodeliverysystems as a mean to target the drug directly to the tumor site. It isnow well established that nanoparticles in the size range 80-120 nm homepreferentially into tumors resulting from the enhanced permeability andretention (EPR) effect (Moghimi, et al., Pharmacol. Rev. (2001), 53(2):283-318). This can reduce systemic side effects and exhibit increasedintratumoral delivery. A nanoliposomal formulation of cisplatin wasfound to deliver 50-200 times more drug to the tumor as compared toadministration of free cisplatin (Harrington, et al. Ann. Oncol. (2001)12: 493-496). Although there was minimal toxicity with the nanoliposomalformulation, it had only modest antitumor activity as compared tocisplatin; reflecting the challenges of not only delivering platinum ina relatively inactive form, but the subsequent need to achievesignificant release and activation within the tumor. A second strategyof encapsulating cisplatin into polymeric systems has been a challengeas a result of its insolubility in organic solvents and partialsolubility in water, which resulted in poor loading or inability tomaintain sustained release. This has required the development ofplatinum (IV) prodrugs that can be modified to increase hydrophobicity,and increase loading in polylactide-polyglycolide copolymernanoparticles (Dhar et al., 2009). Alternatively, cisplatin wasconjugated to N-(2-hydroxypropyl) methacrylamide (HPMA) through peptidylside-chains, and were shown to be biologically active (Lin X, Zhang Q,Rice J R, Stewart D R, Nowotnik D P, Howell S B. Improved targeting ofplatinum chemotherapeutics. The antitumour activity of the HPMAcopolymer platinum agent AP5280 in murine tumour models. Eur J Cancer.2004 January; 40(2):291-7). However, such approaches require processingthrough enzymatic cleavage or intracellular reduction for activation ofthe drug. Similarly, a PAMAM dendrimers-platinum complex, whichincreased drug loading, was found to be 200-550-fold less toxic thancisplatin as a result of strong bonds that are formed between thepolymer and Pt (Haxton K J, Burt H M. Polymeric drug delivery ofplatinum-based anticancer agents. J Pharm Sci. 2009 July;98(7):2299-316).

To engineer a nanoformulation of cisplatin that is facile but overcomesthe challenges associated with current approaches, the inventorsintegrated the existing information on the biotransformation ofcisplatin and understanding of the structure activity relationship thathas emerged through the development of cisplatin analogues. Cisplatingets activated through intracellular aquation of one of the two chlorideleaving groups to form [Pt(NH₃)₂Cl(OH₂)]⁺ and [Pt(NH₃)₂(OH₂)]²⁻,following which the Pt forms covalent bonds to the N₇ position of purinebases to form intrastrand and interstrand crosslinks (Huifang Huang,Leiming Zhu, Brian R. Reid, Gary P. Drobny, Paul B. Hopkins. SolutionStructure of a Cisplatin-Induced DNA Interstrand Cross-Link. Science1995: 270. 1842-1845). In comparison, carboplatin and oxaloplatin, havea cyclobutane-1, l-decarboxylate and an oxalate respectively as theleaving groups, which chelate the platinum more strongly thus conferinggreater stability to the leaving group-PT complex and as a resultexhibit fewer side effects than cisplatin but also lower efficacy thancisplatin (Richard J. Knox, Frank Friedlos, David A. Lydall and John J.Roberts Mechanism of Cytotoxicity of Anticancer Platinum Drugs: EvidenceThat cis-Diamminedichloroplatinum(II) andcis-Diammine-(1,1-cyclobutanedicarboxylato) platinum(II) Differ Only inthe Kinetics of Their Interaction with DNA. Cancer Research 46,1972-1979, Apr. 1, 1986; and Ronald S. Go, Alex A. Adjei. Review of theComparative Pharmacology and Clinical Activity of Cisplatin andCarboplatin. Journal of Clinical Oncology, Vol 17, Issue 1 (January),1999: 409). The inventors selected a 40-mer Poly(isobutylene-alt-maleicacid) (PIMA or PMA) as the polymer because each monomer exhibitsdicarboxylato groups that can be complexed with cisplatin(OH)₂. allowedthe loading of a cisplatin molecule. Furthermore, hydrogenation ofmaleic acid generates succinic acid, which is a component of the Krebscycle. Poly(isobutylene-alt-maleic acid) 2 was synthesized fromPoly(isobutylene-alt-maleic anhydride) 1 by reaction with water in DMFin one step as shown in FIG. 1. Further conjugation of cisplatin toPoly(isobutylene-alt-maleic acid) (PIMA) 2 was achieved by stirringhydrated cisplatin for 48 hours gave PMA-Cisplatin 6. The non-conjugatedcisplatin was removed by dialysis and amount of loading was determinedby NMR and spectrophotometry. Interestingly, the complexation processled to the generation of nanoparticles through a self-assembly process,with the size defined by the number of cisplatin molecules loaded perpolymer. Measurement using dynamic laser light scatter revealed thatsaturating all the complexation sites with cisplatin resulted in a gelformation while loading 15 molecules of cisplatin per polymer resultedin a nanoparticle in the size range of 100 nm. This was validated bytransmission electron microscopy (data not shown).

Cisplatin is a first line therapy for lung cancer, and as a result theinventors studied the effect of PMA-Cisplatin on the viability of Lewislung cancer cells. Treatment with both cisplatin and PMA-cisplatininduced identical cell kill (FIG. 1C). However, PMA also induced tumorcell death. The inventors discovered that this can be overcome throughderivatization of PMA. The inventors derivatized the polymer withethylene diamine under basic conditions (FIG. 2). Interestingly,although the derivatization failed to remove the cytotoxicity of PMA, itincreased the cytotoxicity of the PMA-cisplatin complex. This couldpotentially arise from the fact that the leaving group is less tightlybound as compared to underivatized PMA. Indeed, such an effect has beenseen in the case of carboplatin, which has a lower rate constant foraquation than cisplatin, and as a result is also less cytotoxic. Thenative PMA-cisplatin may be tightly held as compared with PMA-EDAbecause of strong chelation formed by two carboxy groups. To furthermake the polymer more biocompatible the inventors modified the polymerwith glucosamine (GA). PMA-GA-cisplatin was synthesized starting withPMA (1) by reacting with Glucosamine and then with aqueous cisplatin(FIGS. 3 and 11B). All the carrier polymers synthesized were platinatedin aqueous phase at room temperature 25° C. for 2 days, with aquatedcisplatin as platination agent, giving conjugates. At different timepoints, the inventors aliquoted out a small fraction and quantified thetotal loading of cisplatin on the polymer. The inventors observed aloading efficiency of ˜60% by 5 hours of complexation, ˜80% by 30 hoursand 100% by 48 hours of platination. The total drug loaded was 6 mg/15mg of polymer. Aquation of cisplatin was achieved using equimolarcisplatin and AgNO₃ under dark for 48 h. All carriers were routinelyfractionated by dialysis and isolated by freeze-drying for spectroscopiccharacterization. Using DBU resulted in the synthesis of theglucosamine-PMA conjugate as seen in the distinct polymer and sugarpeaks in the NMR results that matches with the predicted NMR values.However, treatments with bases, triethylamine or DIPEA, failed to givethe predicted product, but the NMR traces provided valuable clue todefining the final functional product.

Complexation of cisplatin with PMA-GA resulted in the self assembly ofthe complex into nanoparticles. In certain cases, passing thenanoparticles through a 0.22 micron filter resulted in the generation ofnanoparticles that were in the sub 100 nm range, which is critical forthe particles to home in specifically to the tumor using the EPR effect.Interestingly, cell viability studies revealed that the PMA-GAderivative was devoid of any inherent toxicity to the cells. Incontrast, it retained the efficacy of the aquated Cisplatin (FIG. 4B).Furthermore, derivatization of PMA with polyethylene glycol also removedthe inherent toxicity associated with PMA. Additionally, the same goalcan be achieved by conjugating maleic acid to a polymeric backbone thatis biocompatible.

The increased efficacy with the derivatized chelated polymers ascompared with the native polymer indicates that themonocarboxylato-chelated release drug much easily and showed superioractivity over dicarboxylato-chelated (6). The inventors discovered thatthe polymeric monocarboxylato-chelated platinum compounds represent asizeable advantage over the conjugates in which the metal is bound viadicarboxylic acid. Smooth hydrolytic drug liberation from the carrier inthe monocarboxylato-chelated derivatized PMA conjugates, as compared tothe more retarded hydrolytic fission of the dicarboxylato-chelated inPMA, may explain this enormous difference in cell killing performance.To study this further, the inventors incubated the drug-polymerconjugate with Lewis Lung Cancer cell lysate in a dialysis chamber, andquantified the release of free drug using a calorimetric assay. Theinventors obtained a rapid and sustained release of the active agent(FIG. 4A). It should be noted that the same formulation had beendialyzed in water for 48 hours to remove any free cisplatin and theinventors had obtained 100% loading efficiency, suggesting that theactive agent is not released in neutral conditions but is rapidlyreleased in the presence of tumor cell lysate.

The compositions described herein can be formulated into gels and usedfor sustained released delivery of bioactive agents at specificlocations in a subject. For example, the composition can be used forsustained release delivery of platinum compounds at site of tumors. Insome embodiments, the composition is used for sustain delivery of aplatinum compound after a tumor has been removed.

Pharmaceutical Compositions

For administration to a subject, the polymer linked platinum compoundcan be provided in pharmaceutically acceptable compositions. Thesepharmaceutically acceptable compositions comprise atherapeutically-effective amount of one or more of the platinumcompounds described herein, formulated together with one or morepharmaceutically acceptable carriers (additives) and/or diluents. Asdescribed in detail below, the pharmaceutical compositions of thepresent invention can be specially formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), lozenges, dragees, capsules, pills, tablets(e.g., those targeted for buccal, sublingual, and systemic absorption),boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation; (3) topical application,for example, as a cream, ointment, or a controlled-release patch orspray applied to the skin; (4) intravaginally or intrarectally, forexample, as a pessary, cream or foam; (5) sublingually; (6) ocularly;(7) transdermally; (8) transmucosally; or (9) nasally. Additionally,compounds can be implanted into a patient or injected using a drugdelivery system. See, for example, Urquhart, et al., Ann. Rev.Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. “Controlled Releaseof Pesticides and Pharmaceuticals” (Plenum Press, New York, 1981); U.S.Pat. No. 3,773,919; and U.S. Pat. No. 35 3,270,960.

As used here, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used here, the term “pharmaceutically-acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alchols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in an animal ata reasonable benefit/risk ratio applicable to any medical treatment. Forexample, an amount of a compound administered to a subject that issufficient to produce a statistically significant, measurable change inat least one symptom of cancer or metastasis.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art. Generally, a therapeuticallyeffective amount can vary with the subject's history, age, condition,sex, as well as the severity and type of the medical condition in thesubject, and administration of other pharmaceutically active agents.

As used herein, the term “administer” refers to the placement of acomposition into a subject by a method or route which results in atleast partial localization of the composition at a desired site suchthat desired effect is produced. A compound or composition describedherein can be administered by any appropriate route known in the artincluding, but not limited to, oral or parenteral routes, includingintravenous, intramuscular, subcutaneous, transdermal, airway (aerosol),pulmonary, nasal, rectal, and topical (including buccal and sublingual)administration.

Exemplary modes of administration include, but are not limited to,injection, infusion, instillation, inhalation, or ingestion. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. Inpreferred embodiments, the compositions are administered by intravenousinfusion or injection.

By “treatment”, “prevention” or “amelioration” of a disease or disorderis meant delaying or preventing the onset of such a disease or disorder,reversing, alleviating, ameliorating, inhibiting, slowing down orstopping the progression, aggravation or deterioration the progressionor severity of a condition associated with such a disease or disorder.In one embodiment, at least one symptom of a disease or disorder isalleviated by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, or at least 50%.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patient or subject includes any subset of the foregoing, e.g., all ofthe above, but excluding one or more groups or species such as humans,primates or rodents. In certain embodiments, the subject is a mammal,e.g., a primate, e.g., a human. The terms, “patient” and “subject” areused interchangeably herein. The terms, “patient” and “subject” are usedinterchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models ofdisorders associated with inflammation.

In addition, the methods described herein can be used to treatdomesticated animals and/or pets. A subject can be male or female. Asubject can be one who has been previously diagnosed with or identifiedas suffering from or having a disorder a cancer or metastasis, but neednot have already undergone treatment.

As used herein, the term “cancer” includes, but is not limited to, solidtumors and blood born tumors. The term cancer refers to disease of skin,tissues, organs, bone, cartilage, blood and vessels. The term “cancer”further encompasses primary and metastatic cancers. Examples of cancersthat can be treated with the compounds of the invention include, but arenot limited to, carcinoma, including that of the bladder, breast, colon,kidney, lung, ovary, pancreas, stomach, cervix, thyroid, and skin,including squamous cell carcinoma; hematopoietic tumors of lymphoidlineage, including, but not limited to, leukemia, acute lymphocyticleukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-celllymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma,and Burketts lymphoma; hematopoietic tumors of myeloid lineageincluding, but not limited to, acute and chronic myelogenous leukemiasand promyelocytic leukemia; tumors of mesenchymal origin including, butnot limited to, fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; othertumors including melanoma, seminoma, tetratocarcinoma, neuroblastoma,and glioma; tumors of the central and peripheral nervous systemincluding, but not limited to, astrocytoma, neuroblastoma, glioma, andschwannomas; and other tumors including, but not limited to, xenoderma,pigmentosum, keratoactanthoma, thyroid follicular cancer, andteratocarcinoma. The compounds of the invention are useful for treatingpatients who have been previously treated for cancer, as well as thosewho have not previously been treated for cancer. Indeed, the methods andcompositions of this invention can be used in first-line and second-linecancer treatments.

The compounds of the invention are also useful in combination with knownanti-cancer treatments, including radiation. The methods of theinvention are especially useful in combination with anti-cancertreatments that involve administering a second drug that acts in adifferent phase of the cell cycle, e.g., S phase, than the epothilonesof Formula (1a) or (Ib), which exert their effects at the G2-M phase.

DEFINITIONS

Unless stated otherwise, or implicit from context, the following termsand phrases include the meanings provided below. Unless explicitlystated otherwise, or apparent from context, the terms and phrases belowdo not exclude the meaning that the term or phrase has acquired in theart to which it pertains. The definitions are provided to aid indescribing particular embodiments, and are not intended to limit theclaimed invention, because the scope of the invention is limited only bythe claims. Further, unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean±1%.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The term “comprises”means “includes.” The abbreviation, “e.g.” is derived from the Latinexempli gratia, and is used herein to indicate a non-limiting example.Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

The term “alkyl” refers to saturated non-aromatic hydrocarbon chainsthat may be a straight chain or branched chain, containing the indicatednumber of carbon atoms (these include without limitation methyl, ethyl,propyl, iso-propyl, butyl, 2-methyl-ethyl, t-butyl, allyl, orpropargyl), which may be optionally inserted with N, O, or S. Forexample, C₁-C₆ indicates that the group may have from 1 to 6 (inclusive)carbon atoms in it.

The term “alkenyl” refers to an alkyl that comprises at least one doublebond. Exemplary alkenyl groups include, but are not limited to, forexample, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl and the like.

The term “alkynyl” refers to an alkyl that comprises at least one triplebond.

The term “aryl” refers to monocyclic, bicyclic, or tricyclic aromaticring system wherein 0, 1, 2, 3, or 4 atoms of each ring may besubstituted by a substituent. Exemplary aryl groups include, but are notlimited to, benzyl, phenyl, naphthyl, anthracenyl, azulenyl, fluorenyl,indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like.

The term “cyclyl” or “cycloalkyl” refers to saturated and partiallyunsaturated cyclic hydrocarbon groups having 3 to 12 carbons, forexample, 3 to 8 carbons, and, for example, 3 to 6 carbons, wherein thecycloalkyl group additionally may be optionally substituted. Exemplarycycloalkyl groups include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl,cycloheptyl, cyclooctyl, and the like.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring may be substituted by a substituent. Exemplaryheteroaryl groups include, but are not limited to, pyridyl, furyl orfuranyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl,pyridazinyl, pyrazinyl, quinolinyl, indolyl, thiazolyl, naphthyridinyl,and the like.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring may be substituted by a substituent. Exemplaryheterocyclyl groups include, but are not limited to piperazinyl,pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

The term “optionally substituted” means that the specified group ormoiety, such as an alkyl group, alkenyl group, and the like, isunsubstituted or is substituted with one or more (typically 1-4substituents) independently selected from the group of substituentslisted below in the definition for “substituents” or otherwisespecified.

The term “substituents” refers to a group “substituted” on an alkyl,alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl group atany atom of that group. Suitable substituents include, withoutlimitation, halogen, hydroxy, oxo, nitro, haloalkyl, alkyl, alkenyl,alkynyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino,alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carboxy,hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido,arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano orureido. In some cases, two substituents, together with the carbons towhich they are attached to can form a ring.

As used herein, the term “polymer” refers to the product of apolymerization reaction, and is inclusive of homopolymers, copolymers,terpolymers, tetrapolymers, etc. The term “polymer” is also inclusive ofrandom polymers, block polymers, graft polymers, copolymers, blockcopolymers, and graft copolymers. As used herein, the term “copolymer”refers to polymers formed by the polymerization reaction of at least twodifferent monomers.

The term “copolymer backbone” as used herein refers to that portion ofthe polymer which is a continuous chain comprising the bonds formedbetween monomers upon polymerization. The composition of the copolymnerbackbone can be described in terms of the identity of the monomers fromwhich it is formed without regard to the composition of branches, orsidechains, of the polymer backbone. The term “sidechain” refers toportions of the monomer which, following polymerization, forms anextension of the copolymer backbone.

As used herein, the term “biocompatible” refers to a material that iscapable of interacting with a biological system without causingcytotoxicity, undesired protein or nucleic acid modification oractivation of an undesired immune response. “Biocompatibility” alsoincludes essentially no interactions with recognition proteins, e.g.,naturally occurring antibodies, cell proteins, cells and othercomponents of biological systems.

As used herein an ester sidechains means a sidechains of the formula—R′″C(O)—OR^(E), where RE is independently C1-C6alkyl, C1-C6alkenyl,C1-C6alkynyl, cyclyl, heterocycly, aryl, or heteroaryl, each of whichcan be optionally substituted; and R′″ is a bond or C1-C6 alkylene, werethe alkylene can comprise one or more double or triple bonds and/or thebackbone of the alkylene can be interrupted by O, S, S(O), NH, or C(O).Preferably R′″ is a bond.

As used herein an amide sidechains means a sidechains of the formula—R″C(O)—N(R^(A))₂, where RA is independently H, C1-C6alkyl,C1-C6alkenyl, C1-C6alkynyl, cyclyl, heterocycly, aryl, heteroaryl,saccharide, disaccharide, or trisaccharide, each of which can beoptionally substituted; and R″ is a bond or C1-C6 alkylene, were thealkylene can comprise one or more double or triple bonds and/or thebackbone of the alkylene can be interrupted by O, S, S(O), NH, or C(O).Preferably R″ is a bond.

As used herein a carboxylic acid chain means a sidechains of the formula—R″″C(O)OH where R″″ is a bond or C1-C6 alkylene, were the alkylene cancomprise one or more double or triple bonds and/or the backbone of thealkylene can be interrupted by O, S, S(O), NH, or C(O). Preferably R″″is a bond.

Some non-exhaustive examples of biocompatible polymers includepolyamides, polycarbonates, polyalkylenes, polyalkylene glycols,polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols,polyvinyl ethers, polyvinyl esters, polyvinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andcopolymers thereof, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt,poly(methylmethacrylate), poly (ethylmethacrylate),poly(butylmethacrylate), poly (isobutylmethacrylate),poly(hexlmethacrylate), poly (isodecylmethacrylate),poly(laurylmethacrylate), poly (phenylmethacrylate), poly(methacrylate),poly (isopropacrylate), poly(isobutacrylate), poly (octadecacrylate),polyethylene, polypropylene poly (ethylene glycol), poly(ethyleneoxide), poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinylacetate), poly vinyl chloride, polystyrene, polyhyaluronic acids,casein, gelatin, gluten, polyanhydrides, polyacrylic acid, alginate,chitosan, any copolymers thereof, and any combination of any of these.Additionally, biocompatible polymers and copolymers that have beenmodified for desirable enzymatic degradation, or change upon applicationof light, ultrasonic energy, radiation, a change in temperature, pH,osmolarity, solute or solvent concentration are also amenable to thepresent invention.

The present invention may be defined in any of the following numberedparagraphs:

-   1. A biocompatible conjugated polymer nanoparticle comprising:    -   a copolymer backbone;    -   a plurality of sidechains covalently linked to said backbone;        and    -   a plurality of platinum compounds dissociably linked to said        sidechains.-   2. The nanoparticle of paragraph 1, wherein said plurality of    platinum compounds is selected from Pt(II) compounds, Pt(IV)    compounds, and any combinations thereof.-   3. The nanoparticle of paragraph 1 or 2, wherein at least one of    said plurality of platinum compounds is linked to said sidechain    through at least one coordination bond.-   4. The nanoparticle of paragraph 3, wherein said coordination bond    is between an oxygen of the sidechains and the platinum atom of the    platinum compound.-   5. The nanoparticle of paragraph 4, wherein said oxygen is a    carbonyl oxygen.-   6. The nanoparticle of paragraph 4, wherein said oxygen is an amide    oxygen.-   7. The nanoparticle of any of paragraphs 1-6, wherein said copolymer    comprises maleic acid monomers.-   8. The nanoparticle of paragraph 7, wherein at least one carboxylic    acid of the maleic acid is derivatized to an amide.-   9. The nanoparticle of any of paragraphs 1-8, wherein said copolymer    is poly(isobutylene-alt-maleic acid) (PIMA).-   10. The nanoparticle of any of paragraphs 1-9, wherein said    copolymer comprises from 2 to 100 monomer units.-   11. The nanoparticle of any of paragraphs 1-10, wherein said    copolymer comprises from 25 to 50 monomer units.-   12. The nanoparticle of any of paragraphs 1-11, wherein said    sidechains are selected from the group consisting of polymers,    monosaccharides, dicarboxylic acids, and combinations thereof.-   13. The nanoparticle of any of paragraphs 1-12, wherein said    sidechains are polyethylene glycol (PEG).-   14. The nanoparticle of paragraph 13, wherein said PEG sidechains    have a molecular weight of from 100 to 5000 Dalton.-   15. The nanoparticle of paragraph 13, wherein said PEG sidechains    have a molecular weight of from 1000 to 3000 Dalton.-   16. The nanoparticle of paragraph 13, wherein said PEG sidechains    have a molecular weight of about 2000 Dalton.-   17. The nanoparticle of any of paragraphs 1-12, wherein said    sidechains are monosaccharides.-   18. The nanoparticle of paragraph 17, wherein said monosaccharides    are glucosamine.-   19. The nanoparticle of any of paragraphs 1-18, wherein said    platinum compound is a Pt(II) compound selected from the group    consisting of cisplatin, oxaliplatin, carboplatin, paraplatin,    sartraplatin, and combinations thereof.-   20. The nanoparticle of paragraph 19, wherein said platinum (II)    compound is cisplatin.-   21. The nanoparticle of paragraph 19, wherein said platinum compound    is oxaliplatin.-   22. The nanoparticle of any of paragraphs 1-21, wherein the number    of sidechains corresponds between 50% and 100% of the number of    monomeric units of said polymer backbone.-   23. The nanoparticle of any of paragraphs 1-22, wherein the number    of said sidechains corresponds to a number greater than 90% of the    number of monomeric units of said polymer backbone.-   24. The nanoparticle of any of paragraphs 1-23, wherein the number    of said platinum compounds corresponds between 10% and 100% of the    number of monomeric units of said polymer backbone.-   25. The nanoparticle of any of paragraphs 1-24, wherein the number    of said platinum compounds corresponds between 25% and 75% of the    number of monomeric units of said polymer backbone.-   26. The nanoparticle of any of paragraphs 1-25, wherein said    sidechains comprise dicarboxylic acids.-   27. The nanoparticle of paragraph 26, wherein said dicarboxylic    acids are of the formula HOOC—R—COOH, wherein R is a C₁-C₆alkyl,    C₂-C₆alkenyl, or C₂-C₆alkynyl.-   28. The nanoparticle of paragraph 27, wherein said dicarboxylic acid    is maleic acid.-   29. A biocompatible conjugated polymer nanoparticle comprising:    -   a poly(isobutylene-alt-maleic acid) backbone, wherein said        backbone contains 25 to 50 monomer units;    -   a plurality of PEG sidechains covalently linked to said        backbone, wherein said PEG sidechains have a molecular weight of        from 1000 to 3000 Dalton and wherein the number of said PEG        sidechains corresponds to between 50% and 100% of the number of        monomeric units of said polymer backbone; and    -   a plurality of cisplatin sidegroups dissociably linked to said        backbone wherein the number of said cisplatin sidegroups is        between 25% and 75% of the number of monomeric units of said        polymer backbone.-   30. A biocompatible conjugated polymer nanoparticle comprising:    -   a poly(isobutylene-alt-maleic acid) backbone, wherein said        backbone consist of 40 monomers;    -   a plurality of PEG sidechains covalently linked to said        backbone, wherein said PEG sidechains have a molecular weight of        2000 Dalton and wherein the number of said PEG sidechains is        greater than 90% of monomeric units of said polymer backbone;        and    -   a plurality of cisplatin sidegroups dissociably linked to said        backbone, wherein the number of said cisplatin sidegroups is        between 25% and 75% of the number of monomeric units of said        polymer backbone.-   31. A biocompatible conjugated polymer nanoparticle comprising:    -   a poly(isobutylene-alt-maleic acid) backbone, wherein said        backbone comprises from 25 to 50 monomers;    -   a plurality of glucosamine sidechains covalently linked to said        backbone and wherein the number of said glucosamine sidechains        is between 50% and 100% of monomeric units of said polymer        backbone; and    -   a plurality of cisplatin sidegroups dissociably linked to said        backbone, wherein the number of said cisplatin sidegroups is        between 25% and 75% of the number of monomeric units of said        polymer backbone.-   32. A biocompatible conjugated polymer nanoparticle comprising:    -   a poly(isobutylene-alt-maleic acid) backbone, wherein said        backbone comprises from 25 to 50 monomers;    -   a plurality of glucosamine sidechains covalently linked to said        backbone and wherein the number of said glucosamine sidechains        is greater than 90% of monomeric units of said polymer backbone;        and    -   a plurality of cisplatin sidegroups dissociably linked to said        backbone, wherein the number of said cisplatin sidegroups is        between 25% and 75%, inclusive, of the number of monomeric units        of said polymer backbone.-   33. A carboxylic acid-platinum compound complex conjugated    nanoparticle comprising:    -   a carboxylic acid-platinum compound complex; and    -   a plurality of lipid-polymer chains, wherein the carboxylic acid        portion of said carboxylic acid-platinum compound complex is        covalently bound to said lipid-polymer chains.-   34. The nanoparticle of paragraph 33, wherein the carboxylic acid is    maleic acid.-   35. The nanoparticle of any of paragraphs 33-34, wherein the polymer    is PEG.-   36. The nanoparticle of any of paragraphs 33-35, wherein the    platinum compound is a Pt(II) compound selected from the group    consisting of cisplatin, oxaliplatin, carboplatin, paraplatin,    sartraplatin, and combinations thereof.-   37. The nanoparticle of paragraph 36, wherein the Pt(II) compound is    cisplatin.-   38. The nanoparticle of any of paragraphs 33-37, wherein the    platinum compound loading is from 1%-30%.-   39. The nanoparticle of any of paragraphs 33-38, wherein the    platinum compound loading is from 1%-6%.-   40. A vesicle, micelle, or liposome compound comprising a plurality    of nanoparticles of any of paragraphs 33-39.-   41. A dicarbonyl-lipid compound having the structure

-   42. A vesicle, micelle, liposome or nanoparticle compound comprising    a dicarbonyl-lipid compound of paragraph 41 and a platinum compound,    wherein the platinum compound is dissociably linked to the compound    of paragraph 41.-   43. The nanoparticle of paragraph 42, wherein the platinum compound    is selected from Pt(II) compounds, Pt(IV) compounds, and any    combinations thereof.-   44. The nanoparticle of paragraph 43, wherein said platinum compound    is a Pt(II) compound selected from the group consisting of    cisplatin, oxaliplatin, carboplatin, paraplatin, sartraplatin, and    combinations thereof.-   45. The nanoparticle of paragraph 43, wherein said platinum (II)    compound is cisplatin.-   46. The nanoparticle of paragraph 43, wherein said platinum compound    is oxaliplatin.-   47. A nanoparticle compound comprising a biocompatible polymer,    wherein the polymer comprises at least one monomer having the    formula —CH(CO₂H)—R—CH(C(O)R′)—, wherein R is a bond, C₁-C₆    alkylene, where the alkylene can comprise one or more double or    triple bonds; and R′ is a substituted nitrogen atom. Preferably, R    is a bond.-   48. The nanoparticle of paragraph 47, wherein the polymer comprises    from 2 to 100 monomeric units having the formula    —CH(CO₂H)—R—CH(C(O)R′)—.-   49. The nanoparticle of any of paragraphs 47-48, wherein the polymer    comprises from 25 to 50 monomeric units having the formula    —CH(CO₂H)—R—CH(C(O)R′)—.-   50. The nanoparticle of any of paragraphs 47-49, wherein R′ is

or —NH(CH₂CH₂O)_(m)CH₃, wherein m is 1-150.

-   51. The nanoparticle of any of paragraphs 47-50, further comprising    a bioactive agent.-   52. A pharmaceutical composition comprising:    -   the nanoparticle or compound of paragraphs 1-51; and    -   a pharmaceutically acceptable carrier.-   53. A method of treating cancer or metastasis comprising:    -   administering to a subject in need thereof an effective amount        of the composition of any of paragraphs 1-52.-   54. The method of paragraph 53, wherein said cancer or metastasis is    selected from the group consisting of platinum susceptible or    resistant tumors.-   55. The method of paragraph 54, wherein said cancer or metastasis is    selected from the group consisting of breast, head and neck,    ovarian, testicular, pancreatic, oral-esophageal, gastrointestinal,    liver, gall bladder, lung, melanoma, skin cancer, sarcomas, blood    cancers, brain tumors, glioblastomas, tumors of neuroectodermal    origin and any combinations thereof.-   56. A method of sustain release of a platinum compound at a specific    location in a subject comprising: providing at the location a    composition of paragraph the composition of any of paragraphs 1-52.-   57. The method of paragraph 56, wherein composition is in the form    of a gel.-   58. The method of any of paragraphs 56-57, wherein the location is a    tumor.-   59. The method of paragraph 58, wherein the tumor was removed before    providing the composition.

To the extent not already indicated, it will be understood by those ofordinary skill in the art that any one of the various embodiments hereindescribed and illustrated may be further modified to incorporatefeatures shown in any of the other embodiments disclosed herein.

The following examples illustrate some embodiments and aspects of theinvention. It will be apparent to those skilled in the relevant art thatvarious modifications, additions, substitutions, and the like can beperformed without altering the spirit or scope of the invention, andsuch modifications and variations are encompassed within the scope ofthe invention as defined in the claims which follow. The followingexamples do not in any way limit the invention.

EXAMPLES Materials and Methods

CellTiter 96 AQueous One Solution Cell Proliferation Assay[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assay] reagent was from Promega (Madison, Wis.). AllPolymer solutions were dialyzed in cellulose membrane tubing, typesSpectra/Por 4 and Spectra/Por 6 (wet tubing), with mass-averagemolecular mass cut-off limits of 1000 and 3500 respectively. Operationswere performed against several batches of stirred deionized H2O.Commercially supplied (Sigma, Fluka A G, Aldrich Chemie GmbH) chemicals,reagent grade, were used as received. These includedN,N-Dimethylformamide (DMF), Poly(isobutylene-alt-maleic anhydride),Glucosamine.HCl, mPEG2000NH₂, Diaza(1,3)bicycle[5.4.0]udecane (DBU),Triethyl amine. ¹H NMR and ¹³C NMR were measured at 300 and 400 MHz,respectively, with a Varion-300 or a Brucker-400 spectrometer. 1H NMRchemical shifts are reported as δ values in parts per million (ppm)relative to either tetramethylsilane (0.0 ppm) or deuterium oxide (4.80ppm). Data is reported as follows: chemical shift, multiplicity(s=singlet, d=doublet, t=triplet, q=quartet, m=mutliplet, b=broad),coupling constants (hertz), and integration. Carbon-13 chemical shiftsare reported in ppm relative to CDCl3 (76.9 ppm) or relative to DMSOd₆(39.5 ppm). ¹⁹⁵Pt NMR chemical shifts are reported as δ in ppm relativeto Na₂PtCl₆ (0.0 ppm). In some experiments, ¹H NMR and ¹³C NMR weremeasured at 500 and 125 MHz, respectively, with a Varion 500 or aBrucker-400 spectrometer.

Starting materials were azeotropically dried prior to reaction asrequired, and all air- and/or moisture-sensitive reactions wereconducted in flame- and/or oven-dried glassware under an anhydrousnitrogen atmosphere with standard precautions taken to exclude moisture.

Cell Culture and Cell Viability Assay

The Lewis Lung Carcinoma cell lines (LLC) and Breast Cancer cell line(4T1) were purchased from American Type Culture Collection (ATCC,Rockville, Md., USA). Lewis Lung Carcinoma cells were cultured inDulbecco's Modified Eagle's Medium supplemented with 10% FBS, 50 unit/mlpenicillin and 50 unit/ml streptomycin. The 4T1 cells were cultured inRPMI medium supplemented with 10% FBS, 50 unit/ml penicillin and 50unit/ml streptomycin. Trypsinized cultured LLC and 4T1 cells were washedtwice with PBS and seeded into 96-well flat bottomed plates at a densityof 2×10³ cells in 100 μl of medium. Different concentrations ofconjugates were tested in triplicate in the same 96-well plate for eachexperiment. Medium alone was kept as negative control and CDDP aspositive control. The plates were then incubated for 48 h in a 5% CO₂atmosphere at 37° C. The cells were washed and incubated with 100 μlphenol-red free medium (without FBS) containing 20 μl of the CellTiter96 Aqueous One Solution reagent (Promega, Wis., USA). This assay[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt] (MTS) is a colorimetric method for determining the number ofviable cells in proliferation or cytotoxicity assays. After 2-hincubation in a 5% CO₂ atmosphere at 37° C., the absorbance in each wellwas recorded at 490 nm using a VERSA max plate reader (MolecularDevices, Sunnyvale, Calif., USA).

The absorbance reflects the number of surviving cells. Blanks weresubtracted from all data and results analyzed using Origin software(OriginLab Corporation, Northampton, USA). The mean of triplicatedabsorbance data for each tested dose was divided by the mean ofuntreated control cells. The log of the quotient was used to plot agraph as a function of given dose, i.e. Y=(Tested AbsorbanceMean−Background)/(Untreated Absorbance Mean−Background) vs. X=testeddose.

Particle Size Measurement

High resolution TEM images were obtained on a JEOL 2011 high contrastdigital TEM. Samples were prepared on carbon 300 mesh copper grids(Electron Microscopy Sciences) by adding drops of aqueous nanoparticlesat different concentrations, and allowed to air-dry. The sizedistribution of nanoparticles was studied by dynamic light scattering(DLS), which was performed at 25° C. on a DLS-system (MalvernNanoZetasizer) equipped with a He Ne laser.

Physicochemical Release Kinetics Studies

PIMA-GA-CDDP was suspended in 1 mL of hypoxic-cell lysate from LLC cellline and sealed in a dialysis bag (MWCO˜1000 Da). The dialysis bag wasincubated in 1 mL of PBS buffer at room temperature with gentle shaking.10 □L of aliquot was extracted from the incubation medium atpredetermined time intervals, treated with 90 □L of 1,2-phenylenediamine solution (1.2 mg in 1 mL DMF) and incubated for 3 h at 100° C.The released Pt(IV) was quantified by UV-VIS spectroscopy atcharacteristic wavelength □=704 nm of Pt(IV)-1.2-phenylene diaminecomplex. After withdrawing each aliquot the incubation medium wasreplenished by 10 □L of fresh PBS.

Alternatively, concentrated PIMA-GA-cisplatin conjugate was resuspendedinto 100 μL of double distilled water with pH adjusted to 8.5 or 5.5using IN sodium hydroxide or IN nitric acid and transferred to adialysis tube (MWCO: 1000 KD, Spectrapor). The dialysis tube was putinto a tube containing magnetic pallet and 2 mL solutions of differentpH phosphate-buffered saline. Cisplatin release was studied by gentlystirring the dialysis bag at 300 rpm using IKA stirrer at 25° C. 10 μLaliquots were taken from the outside solution of dialysis membrane bagat predetermined time intervals and subjected to next UV-Vis activecomplex formation reaction by adding 100 μL of ortho-phenyldiamine (1.2mg/ml in DMF) and heating the resulting solution for 3 h. 10 μL of freshsolution was added back to outside solution of dialysis membrane bag tomaintain same volume. The amount of the drug that was released wasevaluated by UV-spectrophotometer (Shimadzu UV 2450) at 706 nm.

FACS Analysis for Apoptosis

Cells were grown in 6-well plates incubated in the presence of cisplatinnanoparticle or free cisplatin at 37° C. for 24 h. After 24 h, the cellswere washed with PBS and collected at 0° C. The cells were then treatedwith annexin V-Alexa Fluor 488 conjugate (Molecular Probes, Invitrogen)and incubated in the dark, at room temperature, for 15 min. The cellswere then washed with PBS and incubated with propidium iodide (PI)solution (50 g/mL; Sigma) containing RNase (1 mg/mL; Sigma). The cellsuspensions were then transferred to FACS tubes and analyzed forAnnexinV/PI staining on a BD FACS Calibur instrument. Data were analyzedusing a CellQuestPro software (BD Biosciences).

Cellular Uptake Studies

LLC and 4T1 cells were seeded on glass coverslips in 24-well plates,50000 cells per well. When cells reached 70% confluency, they weretreated with fluorescein isothiocyanate (FITC)-conjugated cisplatinnanoparticles for different durations of 30 min, 2 h, 6 h, 12 h, and 24h, respectively. For colocalization studies, at indicated time points,the cells were washed with PBS and incubated with Lysotracker Red(Molecular Probes) at 37° C. for 30 min to allow internalization. Thecells were then fixed with 4% paraformaldehyde for 20 min at roomtemperature, then washed twice with PBS and mounted on glass slidesusing Prolong Gold Antifade Reagent (Molecular Probes). Images wereobtained using a Nikon Eclipse TE2000 fluorescence microscope equippedwith green and red filters for FITC and Lysotracker Red, respectively.

In Vivo Murine LLC Lung Cancer and 4T1 Breast Cancer Tumor Model

The LLC Lung Cancer cells and 4T1 Breast Cancer cells (3×105) wereimplanted subcutaneously in the flanks of 4-week-old C57/BL6 and BALB/cmice (weighing 20 g, Charles River Laboratories, MA) respectively. Thedrug therapy was started after the tumors attained volume of 50 mm³. Thetumor therapy consisted of administration of cisplatin nanoparticle andfree cisplatin or oxaliplatin and free oxaliplatin. The formulationswere prepared and validated such that 100 μL of cisplatin nanoparticleand free cisplatin contained 1.25 and 3 mg/kg of cisplatin or 100 μL ofoxaliplatin nanoparticle and free oxaliplatin contained 5 and 15 mg/kgof oxaliplatin Administration was by tail vein injection. PBS (100 μL)administered by tail-vein injection was used as a control for drugtreatment. The tumor volumes and body weights were monitored on a dailybasis. The animals were sacrificed when the average tumor size of thecontrol exceeded 2000 mm³ in the control group. The tumors wereharvested immediately following sacrifice and stored in 100% formalinfor further analysis. All animal procedures were approved by Harvardinstitutional IUCAC.

In Vivo Murine Ovarian Cancer Tumor Model

Ovarian adenocarcinomas were induced in genetically-engineeredK-ras^(LSL/+)/Pten^(fl/fl) mice via intrabursal delivery of adenoviruscarrying Cre recombinase, as described previously. Tumor cells wereengineered to express luciferase once activated by Adeno-Cre, in orderto make tumor imaging feasible before and after drug treatment. Oncemice developed medium to large tumors they were placed into one of fourtreatment groups (controls, cisplatin NP1.25 mg/kg, cisplatin NP-3mg/kg, and free cisplatin), with all drugs administered intravenously(i.v.).

Tumor Imaging and Efficacy Assessment of Drug Treatment

Tumor imaging in vivo was performed with the IVIS Lumina II ImagingSystem. Quantification of bioluminescence was achieved by using theLiving Image Software 3.1 (Caliper Life Sciences). Mice received 150mg/kg of D-luciferin firefly potassium salt via intraperitoneal (i.p.)injection prior to imaging. Five minutes post-luciferin injection,animals were anesthetized in a 2.5% isoflurane induction chamber. Onceanesthesized, mice were placed into the imaging chamber where they werekept under anesthesia by a manifold supplying isoflurane and their bodytemperature was maintained by a 37° C. temperature stage. Bioluminescentsignal was collected fifteen minutes after luciferin administration foran exposure time of thirty seconds. Images were taken a day prior totreatment (day 0, baseline), in the middle of the treatment cycle, andone day following the final treatment. Treatment efficacy was quantifiedby examining the fold increase in bioluminescence of the post-treatmentsignal as compared to baseline. Statistical analysis of the toxicitydata was analyzed using a one-way ANOVA test with the Prism 5™ software.

Biodistribution of Cisplatin

Cisplatin-nanoparticles and free cisplatin were injected i.v. (doseequivalent to 8 mg/Kg of cisplatin) in mice to study its distribution.After 24 hours of injections, the animals were sacrificed and necropsywas performed to harvest the tumor and kidney. In another study, theanimals were dosed repeatedly following the efficacy study protocols,and the animals were sacrificed at the end of the multiple dosing study.The organs were then weighed and dissolved in Conc. HNO₃ (approx. 10 mL)by shaking for 24 hours at room temperature and then heating at 100° C.for 12 hours. To these mixtures were then added 30% H₂O₂, the resultingsolutions were stirred for 24 hours at room temperature and then heatedfor another 12 hours to evaporate the liquids. All solid residues werere-dissolved in 1 mL water and then amount of platinum was measured byinductively coupled plasma-spectrometry (ICP).

Histopathology and TUNEL Assay (Apoptolic Assay)

The tissues were fixed in 10% formalin, paraffin embedded, sectioned andstained with H&E at the Harvard Medical School Core Facility. Tumor andKidney paraffin sections were deparaffinized and stained with standardTMR-red fluorescent terminal deoxynucleotidyl transferase-mediated dUTPnick end labeling (TUNEL) kit following the manufacturer's protocol (InSitu Cell Death Detection Kit, TMR Red, Roche). Images were obtainedusing a Nikon Eclipse TE2000 fluorescence microscope equipped with redfilter.

Toxicity Assessment of Drug Treatment

Body weights were recorded daily to assess toxicity. In addition, liversand spleens were removed at the end of treatment to record weights andperform extensive pathological examination to assess toxicity of vitalorgans. Cell apoptosis in vital organs was measured using TUNNEL assay.Statistical analysis of the toxicity data was performed using a two-wayANOVA test with the Prism 5™ software.

Statistical Analysis

Data were expressed as means±S.D from at least n=3. Statistical analysiswas conducted using the GraphPad Prism software (GraphPad, San Diego,Calif.). The statistical differences were determined by ANOVA followedby Newman Keuls Post Hoc test or Student's t test. p<0.05 wasconsideredtoindicate significant differences.

Example 1 Synthesis of Polymeric Carriers Poly(Isobutylene-Alt-MaleicAcid) PIMA (2)

Poly(isobutylene-alt-maleic anhydride) 1 (1 g) was dissolved in 5 ml ofdry DMF in 10 mL round bottom flask to which was added double distilledwater (1 mL) and then resulting reaction mixture was stirred at 80° C.for 48 h. Solvent was removed under vacuum and low molecular weightimpurities were removed using dialysis. Aqueous polymer solution wasdialyzed for 3 days in cellulose membrane tubing, types Spectra/Por 4and with mass-average molecular mass cut-off limits of 1000. Thecolorless solution was then lyophilized to get 732 mg of white coloredpolymer Poly(isobutylene-alt-maleic acid) PIMA (2). ¹H NMR (300 MHz,D₂O) δ 3.3-3.5 (m), 2.8 (s), 2.6-2.7 (m), 2.5 (s), 2.2-2.3 (m), 0.8-0.9(m).

PIMA-EDA (3)

The 10 mL RB flask equipped with magnetic stirrer and dry nitrogenballoon was charged with Poly(isobutylene-alt-maleic anhydride) PIMA 1(1 g), dry DMF (5 mL), Triethyl amine (0.1 mL) and excess Ethylendiaminedihydrochloride (1 g). The resulting mixture was stirred at 25° C. for48 h. Solvent was removed under vacuum and polymer was purified byremoving low molecular weight impurities such as excess Ethylendiamineusing dialysis bag of molecular cut off of 3.5 KD for 3 days. Thepolymer solution was then lyophilized to get 0.89 g of PIMA-EDA (3).¹H-NMR (300 MHz, D₂O) δ 3.1-3.2 (m), 2.9-3.0 (m), 2.6-2.8 (m), 2.5 (m),0.8-1.0 (m).

PIMA-GA Polymer (4)

Poly(isobutylene-alt-maleic anhydride) PIMA 1 (0.0064 g 0.001 mmol) wasdissolved in DMF (5 mL) and then was added DBU (0.032 mL, dissolved in 1mL dry DMF, 0.21 mmol) and the mixture was stirred at 25° C. for 1 h. Tothis solution was added Glucosamine (0.046 g 0.21 mmol) directly. Theresulting reaction mixture was allowed to stir at room temperature for48 h and then quenched by adding double distilled water (1 mL). Theorganic solvent was evaporated under vacuum for 12 hours. The resultingpale yellow solid was purified by dialysis for 3 days using dialysis bagsupplied by Pierce (Thermoscientific) of molecular cut off of 3.5 KD tocolorless solution. Lyophilization gave 104 mg of white colored PIMA-GA(4) polymer. ¹H-NMR (300 MHz, CDC¹³) δ 7.54-7.65 (m, 2H), 7.33-7.45 (m,2H), 7.02-7.19 (m, 14H), 6.93-6.97 (m, 2H), 6.83-6.89 (m, 2H), 6.55 (s,2H), 6.15-6.19 (m, 2H), 3.90 (s, 2H), 3.58 (s, 6H). ¹H-NMR (300 MHz,D₂O) δ 8.2-8.3 (m), 7.0-7.1 (m), 5.0-5.1 (m), 3.0-3.9 (m), 2.1-2.3 (m),1.1-1.9 (m), 0.7-1.0 (m).

In another experiment, PIMA (0.045 g) was dissolved in DMF (5 mL) andthen was added solution of DBU (0.23 mL) and glucosamine (0.323 gdissolved in 5 mL dry DMF). The resulting reaction mixture was allowedto stir at room temperature for 48 h and then quenched by adding ddwater (1 mL). The organic solvent was evaporated under vacuum. Theresulting pale yellow solid was purified by dialysis for 3 days usingdialysis bag of molecular cut off of 3.5 KD. Lyophilization gave 104 mgof slightly yellow colored PIMA-GA polymer. ¹H-NMR (300 MHz, D₂O) δ8.2-8.3 (m), 7.07.1 (m), 5.0-5.1 (m), 3.0-3.9 (m), 2.1-2.3 (m), 1.1-1.9(m), 0.7-1.0 (m).

PIMA-PEG Polymer (5)

The Poly(isobutylene-alt-maleic anhydride) PIMA 1 (3 mg, 0.0005 mmol)and DBU (0.0023 mL, 0.015 mmol) was dissolved in Dry DMF (10 mL) in 25mL RB flask under N₂ for 1 h and then was added PEG-NH₂ (20 mg, 0.01mmol), the resulting reaction solution was then heated at 80° C. withcontinuous stirring for 3 days. The reaction was allowed to cool to roomtemperature and then water (1 mL) was added and continue stirring for 1h. Solvents are removed under vacuum and unreacted PEG-NH₂ of MW 2 KDwas removed from required polymer by dialysis. Dialysis was carried outfor 5 days using membrane of molecular cut off of 3.5 KD supplied byPierce (Thermoscientific) to give colorless solution which was thenlyophilized to give 19 mg white colored PIMA-PEG (5). ¹H-NMR (300 MHz,D₂O) δ 3.5-3.7 (m), 3.0-3.1 (m), 2.5-2.8 (m), 0.7-1.0 (m).

Example 2 Synthesis of Conjugates Aquation of CDDP

CDDP (30 mg) and AgNO₃ (17 mg) was added to 10 ml double distilledwater. The resulting solution was stirred in dark at room temperaturefor 24 h. AgCl precipitates were found after reaction. AgCl precipitatesare removed from reaction by centrifugation at 10000 rpm for 10 min. Thesupernatant was further purified by passing through 0.2 am filter.

PIMA-CDDP (6)

Poly(isobutylene-alt-maleic acid) PIMA 2 (0.006 g, 0.001 mmol) wasdissolved in 1 ml double distilled water containing CDDP (0.00084 g,0.0028 mmol) in 10 mL round bottom flask to and then resulting reactionmixture was stirred at room temperature (25° C.) for 48 h. The PIMA-CDDP(6) conjugate was further purified by dialyzing it in cellulose membranetubing, types Spectra/Por 4 and with mass-average molecular mass cut-offlimits of 1000. The resulting turbid solution was then lyophilized toget white colored PIMA-CDDP (6) conjugate. The conjugate wasre-suspended for cell culture experiments.

PIMAA-EDA-CDDP (7)

In 10 mL RB flask was weighed PIMA-EDA 3 (0.007 g, 0.001 mmol) polymerto which was added CDDP (0.0084 g, 0.0028 mmol) dissolved in doubledistilled water (1 mL). The solution was then stirred at roomtemperature (25° C.) for 48 h. Dialysis using cellulose membrane withmolecular mass cut-off limits of 1000 and lyophilization gave yellowishcolored PIMA-EDA-CDDP (7) conjugate.

PIMAA-GA-CDDP (8)

To PIMA-GA 4 (0.0036 g, 0.0003 mmol) weighed in 10 mL RB flask equippedwith magnetic stirrer was added 1 ml double distilled water containingCDDP (0.001 g, 0.0033 mmol) and then the solution was stirred at roomtemperature (25° C.) for 48 h. The PIMA-GA-CDDP (8) conjugate formed insolution was further purified by dialysis to remove unattached CDDP withmass-average molecular mass cut-off limits of 1000 for 2-3 hours.Lyophilization of the dialyzed solution resulted in slightly yellowcolored PIMA-GA-CDDP (8) conjugate.

PIMA-PEG-CDDP (9)

The brush polymer PIMA-PEG 5 (0.019 g, 0.00007 mmol) was taken in 10 mLRB flask mixed with CDDP (0.0002 g, 0.0007 mmol) dissolved in 0.3 mLdouble distilled water. After stirring for 3 days at room temperature(25° C.) the resulting turbid reaction mixture was dialyzed. Thesolution containing PIMA-PEG-CDDP (2) conjugate was further purified bydialyzing it in cellulose membrane tubing, types Spectra/Por 4 and withmass-average molecular mass cut-off limits of 1000 for 2-3 hours toremove free CDDP. PIMA-PEG-CDDP (9) conjugate was then lyophilized toget white colored solid. The conjugate was re-suspended in doubledistilled water for cell culture experiments.

FITC-Labeled PIMA-GA-CDDP

Poly(isobutylene-alt-maleic anhydride) PIMA (0.006 g) was dissolved inDMF (5 mL) and then was added a solution of DBU (0.0053 mL in DMF) andGlucosamine (0.0075 g dissolved in 5 mL dry DMF) the mixture was stirredat 25° C. for 1 h. The resulting reaction mixture was allowed to stir at25° C. for 48 h and then to which was added 0.0022 g FITC-EDA (FITC-EDAwas synthesized by stirring Fluorescein isothiocyanate in excessethylene diamine at 25° C. for 12 h in DMSO) and continue stirring foranother 12 h, reaction mixture was quenched by adding double distilledwater (1 mL). The organic solvent was evaporated under vacuum. Theresulting orange solid was purified by dialysis for 3 days usingdialysis bag of molecular cut off of 3.5 KD. Lyophilization gavefluorescent orange PIMA-GA-FITC polymer. To this FITC labeled polymer(PIMA-GA-FITC, 0.004 g) was added 1 ml double distilled water containingcisplatin (0.001 g) and then the solution was stirred at roomtemperature (25° C.) for 48 h. The PIMA-GA-FITC-cisplatin conjugateformed in solution was further purified by dialysis to remove unattachedcisplatin with mass-average molecular mass cut-off limits of 1000.Lyophilization of the dialyzed solution resulted in orange colored FITClabeled PIMA-GAFITC-cisplatin conjugate nanoparticles.

PIMA-Oxaliplatin

Poly(isobutylene-alt-maleic acid) (PIMA) (6 mg) was dissolved in 1 mldouble distilled water containing oxaliplatin-OH (1 mg) in a roundbottom flask to and then resulting reaction mixture was stirred at roomtemperature (25° C.) for 48 h. The PIMA-oxaliplatin conjugate wasfurther purified by dialyzing it in cellulose membrane tubing, typesSpectra/Por 4 and with mass-average molecular mass cut-off limits of1000. The resulting turbid solution was then lyophilized to getPIMA-oxaliplatin conjugate. The conjugate was re-suspended for cellculture experiments.

PIMA-GA-Oxaliplatin

To PIMA-GA (12 mg) weighed in 10 mL RB flask equipped with magneticstirrer was added 1 ml double distilled water containing oxaliplatinOH(1 mg) and then the solution was stirred at room temperature (25° C.)for 48 h. The PIMA-GA-oxaliplatin conjugate formed in solution wasfurther purified by dialysis to remove unattached oxaliplatin withmass-average molecular mass cut-off limits of 1000. Lyophilization ofthe dialyed solution resulted in yellow colored PIMA-GA-oxaliplatinconjugate.

Example 3 NMR Analysis of PIMA-GA Polymer Synthesis Using DifferentBases Synthesis of PIMA-GA Using DBU as the Base

Glucosamine hydrochloride (360 mg, 1.66 mmol, 200 equiv) was suspendedin 5 mL DMF and treated with DBU (250 □L, 1.66 mmol, 200 equiv) at roomtemperature for 1 h. After 1 h glucosamine/DBU (in DMF) solution wasadded drop wise into poly (isobutylene-alt-maleic anhydride) (50 mg,0.008 mmol, 1 equiv) solution in 5 mL DMF and the reaction mixture wasstirred for 72 h at room temperature. The reaction mixture was quenchedwith 3 mL of dd-water. The PIMA-GA conjugate was purified by dialysisusing 2000 MWCO dialysis bag for 72 h. The product was lyophilized for48 h to obtain 100 mg cream yellow powder. The product was characterizedby ¹H NMR spectroscopy (300 MHz). Solubility: product was soluble inwater but not soluble in organic solvent e.g. acetone, methanol oracetonitrile. ¹H NMR (300 MHz): □ (ppm)=5.2-5.3 (m, 0.14 H, sugarproton), 5.0-5.1 (m, 0.4 H, sugar proton), 3.6-4.0 (m, 13.07 H, sugarproton), 3.25-3.5 (m, 15.48 H, sugar proton), 3.0-3.2 (m, 6.98 H, sugarproton), 2.5-2.6 (m, 6.97 H, PIMA proton), 1.4-1.7 (m, 19.86 H, PIMAproton), 0.7-1.2 (m, 23.77 H, PIMA proton). Total sugar protons: totalPIMA protons=36.07:50.6=0.71. This fits well with the predictedstructure if all the residues are derivatized sugar protons and PIMAprotons in PIMA-GA conjugate monomer.

Synthesis of PIMA-GA Using Diisoproylethylamine (DIPEA) as Base

Glucosamine hydrochloride (179 mg, 0.83 mmol, 100 equiv) was suspendedin 2 mL DMF and treated with DIPEA (145 □L, 0.83 mmol, 100 equiv) atroom temperature for 1 h. After 1 h poly (isobutylene-alt-maleicanhydride) (50 mg, 0.008 mmol, 1 equiv) (dissolved in 3 mL DMF) wasadded into the reaction mixture and stirred for 24 h at roomtemperature. The reaction mixture was quenched with 3 mL of dd-water.The PIMA-GA conjugate was purified by dialysis using 1000 MWCO dialysisbag for 24 h. The product was lyophilized for 48 h to obtain 106 mgwhite powder. The product was characterized by ¹H NMR spectroscopy (300MHz). Solubility: product was soluble in water but not soluble inorganic solvent e.g. acetone, methanol or acetonitrile. ¹H NMR (300MHz): □ (ppm)=5.2-5.3 (m, 0.4 H, sugar proton), 4.9-5.1 (m, 2.0 H, sugarproton), 3.4-3.6 (m, 21.86 H, sugar proton), 3.2-3.3 (m, 6.16 H, sugarproton), 2.9-3.1 (m, 3.81 H, sugar proton), 2.4-2.7 (broad, 4.39 H, PIMAproton), 2.1-2.4 (broad, 4.54 H, PIMA proton), 1.7-2.0 (broad, 3.13 H,PIMA proton), 1.3-1.5 (broad, 1.58 H, PIMA proton), 1.1-1.2 (m, 24.12 H,PIMA proton), 0.6-0.9 (m, 27.94 H, PIMA proton). Total sugar protons:total PIMA protons=39.21:61.11=0.64.

Synthesis of PIMA-GA Using Trietylamine as Base

Glucosamine hydrochloride (143 mg, 0.66 mmol, 80 equiv) was suspended in2 mL DMF and treated with triethylamine (100 □L, 0.66 mmol, 80 equiv) atroom temperature for 1 h. After 1 h poly (isobutylene-alt-maleicanhydride) (50 mg, 0.008 mmol, 1 equiv) was added into the reactionmixture and stirred for 24 h at room temperature. The reaction mixturewas quenched with 3 mL of dd-water. The PIMA-GA conjugate was purifiedby dialysis using 1000 MWCO dialysis bag for 24 h. The product waslyophilized for 48 h to obtain 100 mg white powder. The product wascharacterized by ¹H NMR spectroscopy (300 MHz). Solubility: product wassoluble in water but not soluble in organic solvent e.g. acetone,methanol or acetonitrile. ¹H NMR (300 MHz): □ (ppm)=5.2-5.3 (m, 0.44 H,sugar proton), 4.9-5.1 (m, 1.51H, sugar proton), 3.7-3.8 (m, 19.01 H,sugar proton), 3.3-3.4 (m, 6.43 H, sugar proton), 3.1-3.2 (m, 11.82,sugar proton), 2.93-2.94 (m, 2.23 H, PIMA proton), 2.6-2.7 (m, 5.84 H,PIMA proton), 2.2-2.5 (broad, 4.91 H, PIMA proton), 1.8-2.1 (broad, 3.83H, PIMA proton), 1.4-1.6 (broad, 2.52 H, PIMA proton), 1.8-1.2 (m, 18.04H, PIMA proton), 0.9-1.0 (m, 23.77 H, PIMA proton). Total sugar protons:total PIMA protons=34.31:65.7=0.52.

Example 4 Time Dependent Loading Efficiency of PIMA-GA-CDDP

Method: PIMA-GA conjugate (50 mg, 0.004 mmol) was dissolved in 1 mLdd-water followed by the addition of (NH₂)₂Pt(OH)₂ (3 mL, 0.057 mmol).The reaction was stirred at room temperature for 48 h. 200 □L ofaliquots were taken out from the reaction mixture after eachpre-determined time points (5 h, 31 h and 48 h). The aliquots werefiltered through Microcon centrifugal filter device having regeneratedcellulose membrane of 3000 MWCO to separate the PIMA-GA-CDDP conjugate.The polymer was washed thoroughly (200 □L×2) with dd-water to remove anyplatinum reagent. The platinum content in polymer was determined by themethod described before.

Result: The change in Pt-loading efficiency in PIMA-GA conjugate wasdetermined by the ability of conjugating 1,2-phenylenediamine with Ptgiving rise to UV-VIS spectra at wavelength □=706 nm. Neither thepolymer nor 1,2-phenyldiamine has any characteristic peak at thiswavelength. How the Pt-content changes with time in the reaction betweenPIMA-GA and hydroxy-platin was monitored by UV-VIS spectra. At differentpre-determined time points (5 h, 31 h and 48 h) 200 n L of aliquots weretaken out from the reaction mixture and the Pt-loading in the polymerconjugate was determined. FIG. 9 shows that loading of platinum in thepolymer conjugate increases with time from 190 □g/mg (5 h) to 210 □g/mg(31 h) and reaches maximum 347 □g/mg at 48 h. This indicates almost 100%of the Pt is complexed with the polymer at this time point as themaximal predicted loading is 37.5% per polymer unit, and we attain 34.7%Pt per polymer.

Example 5 Rational Optimization of the Polymer Based onStructure-Activity Relationship

In order to improve efficacy of the nanoparticles, the inventorsderivatized one arm of each monomer of the polymer with biocompatibleglucosamine to generate PIMA-glucosamine conjugate (PIMA-GA) (FIG. 11B).This converted the dicarboxylato bonds with Pt to a monocarboxylato bondand a coordinate bond, which can release Pt more easily given that acoordinate bond is less stable than a monocarboxylato linkage (FIG.11B).

Nuclear magnetic resonance (NMR) characterization of the Pt environmentrevealed that complexation of PIMA-GA and cisplatin in an acidic pH (pH6.5) generated an isomeric state [PIMA-GA-Cisplatin (O->Pt)] (8)characterized by the monocarboxylato and a O->Pt coordination complex ascharacterized by a single Pt NMR peak at −1611.54 (FIG. 11B).Interestingly, complexing the cisplatin with PIMA-GA at an alkaline pH(pH 8.5) favored the formation of an isomeric PIMA-GA-Cisplatin (N->Pt)complex (10), where the Pt is complexed through a monocarboxylato and amore stable N->Pt coordinate bond characterized by a unique peak at−2210 (FIG. 11B). Excitingly, the existing of these two pH-dependentstates allowed the inventors to further dissect the impact of Ptenvironment, specifically the leaving groups, on the biologicalefficacy.

The complexation of cisplatin to PIMA-glucosamine (PIMA-GA) polymer at aratio of 15:1 resulted in self assembly into nanoparticles in thedesired narrow size bandwidth of 80-150 nm as confirmed byhigh-resolution transmission electron microscopy (data not shown) andDLS (FIG. 12A). Furthermore, the inventors achieved a loading of 175±5μg/mg of polymer (FIG. 12B), which is significantly higher than can beachieved using traditional nanoparticle formulations (Avgoustakis K,Beletsi A, Panagi Z, Klepetsanis P, Karydas A G, Ithakissios D S.PLGA-mPEG nanoparticles of cisplatin: in vitro nanoparticle degradation,in vitro drug release and in vivo drug residence in blood properties. JControl Release. 2002 Feb. 19; 79(1-3):123-35).

Example 6 Characterizing the Uptake and Efficacy of Nanoparticles InVitro

Tagging the polymer with fluorescein (FIG. 15) enabled the temporaltracking of uptake of the nanoparticles into the cells, which wereco-labeled with a lysotracker-red dye to label the endolysosomalcompartments. A rapid uptake of the nanoparticles was observed in theLLC cells within 15 min of treatment with internalization into theendolysosomal compartment as evident by colocalization of theFITC-nanoparticles and the Lysotracker-Red dye (data not shown). Incontrast, the uptake into 4T1 cells was delayed, with internalizationinto the endolysosomal compartment evident only after 2 hourspost-incubation. Over a 12 hour period, the fluorescent signals from thelysosomal compartment and the FITC-conjugated dissociate, suggesting acytosolic distribution of the polymer after processing within thelysosome (data not shown).

To test the efficacy of the PIMA-GA-cisplatin nanoparticles in vitro,the inventors performed cell viability assays using Lewis lung carcinoma(LLC) and 4T1 breast cancer cell lines. Cell viability was quantifiedusing a MTS assay at 48 hours post-incubation. Interestingly, the LLCcells (FIG. 13A) were more susceptible to cisplatin-nanoparticles thanthe 4T1 breast cancer cells (FIG. 13B). Excitingly, PIMA-GA-cisplatin(O->Pt) nanoparticles (8) demonstrated significant LLC cell kill withIC50 values (4.25±0.161M) similar (P>0.05) to cisplatin (IC50=3.87±0.37μM), and superior to carboplatin (IC50=14.75±0.38 μM), which supportsthe hypothesis that the rate of aquation is critical for efficacy (FIG.13A-F). A similar efficacy was observed when the inventors replacedglucosamine with ethylene diamine, which creates a similar Ptcomplexation environment as glucosamine (FIG. 13A). This wasadditionally supported by the observation that PIMA-GA-cisplatin (N->Pt)nanoparticles (IC50=6.36±0.19 μM) were significantly less active thancisplatin, suggesting that the platinum environment is critical indefining the rate of aquation. To further validate the role ofcomplexation environment, the inventors generated PIMA-GA(20), whereonly 20 of the 40 monomers comprising a PIMA polymer were derivatizedwith glucosamine, thereby introducing dicarboxylato bonds and reducingthe monocarboxylato plus coordinate bonds that complex Pt to PIMA-GA. Asshown in FIG. 13F, the concentration-efficacy curve shifts to the rightwith PIMA-GA(20)-cisplatin (EC50=5.85±0.13 μM) as compared withPIMA-GA-cisplatin (O->Pt) nanoparticles, where all the 40 monomers arederivatized with glucosamine. Empty PIMA-GA polymer had no effect on thecell viability. Table 1 summarizes the EC50 values.

As shown in FIG. 13A, while the polymer alone induced cell death at thehighest concentrations, complexation of cisplatin significantly shiftedthe concentration-effect curve to the left, indicating that thePIMA-cisplatin nanoparticle induces cell kill. However, even at aconcentration of 50 uM, the PIMA cisplatin failed to induce completecell kill. In contrast, cisplatin exerts complete cell kill at aconcentration greater than 20 uM. This reduction in the efficacy ofpalatinate when complexed with PIMA can be explained by thedicaroboxylateo linkage between the platinum and the maleci acidmonomers, which tightly binds the Pt similar to the linkage that existsin carboplatin, which similarly is less efficacious than cisplatin.

Labeling the cells for expression of phosphatidylserine on the cellsurface, revealed that the cisplatin treatments could induce apoptoticcell death, with LLCs being more susceptible than 4T1 cells (FIG.14A-14J).

TABLE 1 EC50 values for various complexes EC50 (μM) PIMA30:PIMA-GA-Cisplatin [acidic] 5.29 ± 0.11 PIMA30: PIMA-GA-Cisplatin [basic]6.84 ± 0.14 Cisplatin 3.87 ± 0.37 Carboplatin 14.75 ± 0.38  PIMA40-200:PIMA-GA-Cisplatin [acidic] 4.25 ± 0.16 PIMA40-200: PIMA-GA-Cisplatin[basic] 6.36 ± 0.19 PIMA-GA20-Cisplatin [acidic] 5.85 ± 0.13

Example 7 The Release of Active Cisplatin from Nanoparticle ispH-Dependent

Given that the nanoparticles localized to the lysosomal compartment, theinventors tested the release of Pt from the nanoparticles at pH 5.5,mimicking the acidic pH of the endolysosomal compartment of the tumor(Lin, et al., Eur. J. Cancer, 2004 40(2):291-297). The inventors alsoselected pH8.5 as a reference pH in the alkaline range. As shown in FIG.16, at pH5.5 PIMA-GA-cisplatin (O->Pt) nanoparticles resulted in asustained but significant release of cisplatin monitored over a 70 hourperiod. In contrast the release at pH8.5 was significantly lower,indicating a pH-dependent release of Pt. Interestingly,PIMA-GA-cisplatin (N->Pt) released significantly lower amounts of Pteven at pH5.5, consistent with the fact that the N->Pt coordinate bondis stronger than the O->Pt linkage. As expected, the inventors observedthat PIMA-cisplatin nanoparticles exhibited significantly lower rates ofPt release as compared with both PIMA-GA-cisplatin (N->Pt) andPIMA-GA-cisplatin (O->Pt) as the Pt is held by more stable dicarboxylatobonds instead of a monocarboxylato and a coordinate bond.

Example 8 Nanoparticle Induces Tumor Growth Delay and Regression withReduced Nephrotoxicity

As PIMA-GA-cisplatin (O->Pt) nanoparticles exhibited the desired releaserates for platinum and also exhibited in vitro efficacy comparable tocisplatin, the inventors validated the therapeutic efficacy of thenanoparticles in vivo. They randomly sorted mice bearing establishedLewis lung carcinoma or 4T1 breast cancer into five groups respectivelyand treated each group with three doses of (i) PBS (control); (ii)Cisplatin (1.25 mg/kg); (iii) Cisplatin (3 mg/kg); (iv)PIMA-GA-Cisplatin (O->Pt) nanoparticles (1.25 mg/kg); (v)PIMA-GA-Cisplatin (O->Pt) nanoparticles (3 mg/kg). The mice injectedwith PBS formed large tumors by day 16 (day after the last injection),and consequently, were euthanized. The animals in the other groups werealso sacrificed at the same time point to evaluate the effect of thetreatments on tumor pathology. As shown in FIG. 5A-F, cisplatin induceddose-dependent tumor inhibition, and at a dose equivalent to 1.25 mg/kgof cisplatin, administration of the nanoparticle formulation resulted ingreater inhibition of lung carcinoma progression as compared with thefree drug. However, at a dose equivalent to 3 mg/kg, free cisplatinresulted in a significant reduction in body weight indicating systemictoxicity. In contrast, animals treated with nanoparticles equivalent to3 mg/kg of cisplatin exhibited weight gain, although tumor inhibitionwas similar in both treatment groups (data not shown). Furthermore,necropsy revealed that treatment with free cisplatin resulted in asignificant reduction in the weights of kidney and spleen (FIGS. 5D and5E), indicating nephrotoxicity and hematotoxicity consistent withprevious reports. Excitingly, cisplatin nanoparticles had no effect onthe weights of the kidneys, and reduced spleen size only at the highestdose (FIGS. 5D and 5E). This was further validated by pathologicalanalysis of kidney H&E stained cross-sections, which revealedsignificant tubular necrosis in the animals treated with free cisplatinas compared with cisplatin nanoparticle. To elucidate the mechanismunderlying tumor inhibition, the inventors labeled the tumor crosssections for TUNEL, which revealed a significant induction of apoptosisfollowing treatment with both free cisplatin and PIMA-GAcisplatin(O->Pt)nanoparticles (data not shown). Interestingly, labeling the kidneysections for TUNEL demonstrated significant apoptosis in the animalstreated with free cisplatin as opposed to minimal nephrotoxicity in thenanoparticle-treated group (data not shown). Indeed, biodistributionstudies using inductively coupled plasma-spectrometry (ICP) revealedthat the concentration of Pt in the kidney following administration ofthe cisplatin nanoparticle is 50% of that attained followingadministration of free drug (FIG. 5F), which can explain the reductionin nephrotoxicity.

Treatment with cisplatin (1.25 mg/kg) exhibited only a minor tumorgrowth inhibition as compared with control; in contrast, treatment withnanoparticle-cisplatin at the same dose exerted a dramatic increase inthe antitumor efficacy (FIG. 5A). This is consistent with the fact thatnanoparticles enable a significantly higher concentration of the activeagent to be attained within the tumor as compared to free drug²⁰. At thehigher dose, both the free drug and the nanoparticle achieved similarantitumor efficacy (FIG. 5A), which is potentially the theoretical limitof the drug. However, the free drug at this dose resulted in a greaterthan 20% loss of body weight (FIG. 5B), which is an indicator ofnon-specific toxicity. Indeed, it induced significant nephrotoxicity asseen by the loss of weight of the kidney (FIG. 5D). Furthermore,although the blood counts were not different between the varioustreatment groups (FIG. 5C), there was significant loss of weight of thespleen at the highest dose of the free cisplatin (FIG. 5E). In contrast,nanoparticle-cisplatin exhibited no such toxicity even at the highestdose, open up the possibility of dosing at higher levels or for longertime periods, both of which can dramatically impact antitumor outcomes.Furthermore, the ease of manufacturing, the low costs of materials andthe increase in therapeutic efficacy and reduction of toxicity canbecome an example of nanotechnology impacting global health. Withoutwishing to be bound by theory, the increased therapeutic index can arisefrom a preferential accumulation of the nanoparticles in the tumorsarising from the well-studied EPR effect, and circumventing the kidneyas it exceeds the size limit for clearance, which in a previous studywas shown to be less than 5 nm (Choi H S, Liu W, Misra P, Tanaka E,Zimmer J P, Itty Ipe B, Bawendi M G, Frangioni J V. Renal clearance ofquantum dots. Nat Biotechnol. 2007, 25:1165-70).

Both free cisplatin and PIMA-GA-cisplatin(O->Pt) nanoparticles resultedin similar levels of tumor growth inhibition in the 4T1 breast cancermodel (FIG. 17A-17D). Interestingly, both 1.25 mg/kg and 3 mg/kg freecisplatin induced a significant loss of body weight as compared with thecisplatin-nanoparticle treated groups. Consistent with the observationsin the lung cancer model, while free cisplatin induced significantapoptosis in the kidney, the nanoparticle-cisplatin treated groupsexhibited minimal apoptosis in the kidney but significant levels ofapoptosis in the tumor.

In addition to lung and breast cancer models, the inventors furtherevaluated the PIMA-GA-cisplatin(O->Pt) nanoparticle in an ovarian cancermodel. Epithelial ovarian cancer is the deadliest malignancies of thefemale reproductive cycle. The discovery of frequent somatic PTENmutations and loss of heterozygosity at the 10q23 PTEN locus inendometrioid ovarian cancer implicates a key role for PTEN in theetiology of this epithelial ovarian cancer subtype (Obata, K. et al.Frequent PTEN/MMACl mutations in endometrioid but not serous or mucinousepithelial ovarian tumors. Cancer Res. 58, 2095-2097 (1998) and Sato, etal., Cancer Res. 2000, 60: 7052-7056 and Sato, et al., Cancer Res. 2000,60: 7052-7056). Similarly, K-RAS oncogene is also mutated inendometrioid ovarian cancer, albeit at a lesser frequency (Cuatrecasas,et al., Cancer (1998) 82:1088-1095). In a recent study, the combinationof these two mutations in the ovarian surface epithelium was found toinduce invasive and widely metastatic endometrioid ovarianadenocarcinomas with complete penetrance, making it a good model formimicking human tumor progression. In this transgenic modelvehicle-treated animals exhibited rapid tumor progression as quantifiedby luciferase expression. Treatment with the cisplatin nanoparticlesresulted in a dose-dependent inhibition of tumor progression, with thelower dose equivalent to 1.25 mg/kg exerting a similar inhibition as a 3mg/kg dose of free cisplatin (FIG. 18A). Treatment with the higher doseof cisplatin-nanoparticle (equivalent to 3 mg/kg of cisplatin) resultedin greater tumor inhibition without any significant loss of body weightas observed with an equidose of free cisplatin, which is approved forclinical use in ovarian cancer (FIG. 18B). Furthermore, TUNEL stainingrevealed significant apoptosis in the kidney at 3 mg/kg of freecisplatin while the cisplatinnanoparticles at equivalent Ptconcentration did not induce apoptosis of the nephrons.

Example 9 Biodistribution of Cisplatin-Nanoparticles Following MultipleDosing

To study the biodistribution of the cisplatin nanoparticles, theinventors harvested the tumors at the end of the multiple-dosingexperiments, where each animal received three doses of free drug or thecisplatin nanoparticle. As shown in FIGS. 19A-19B, there was apreferential accumulation Pt in both breast and ovarian tumors whenadministered as a nanoparticle as opposed to when delivered as freecisplatin.

Example 10 Toxicity Assessment of Treatment with PIMA-GA-Oxaliplatin

As seen in FIG. 23, at a dose of 15 mg/kg of free oxaliplatin, all theanimals died due to systemic toxicity. In contrast no toxicity wasevident even at this dose in the case of oxaliplatin nanoparticle.

Example 11 Synthetic Scheme of Lipid-Cisplatin Conjugate

In addition to the PMA-GA-cisplatin conjugate, the inventors have alsoengineered an analog, where maleic acid is conjugated to PEG end of apegylated lipid (PEG2000-DSPE). The inventors complexed Pt to the maleicacid, resulting in the formation of a platinated lipid derivative wherethe Pt is at the hydrophilic end and the lipid forms the hydrophobicend. These form micelles in water, and the loading efficiency is 45μg/mg of lipid derivative. This can be increased by using a lowermolecular weight PEG or lipid. See FIG. 10.

Example 12 Cisplatin-Liponanoparticles Materials and Method

All reactions were performed under inert conditions unless otherwiseindicated. All commercially obtained compounds were used without furtherpurification. DCM, dry DCM, Methanol, Cholesteryl Chloroformate,Cholesterol, Ethylenediamine, Succinic Anhydride, Silver Nitrate, SodiumSulphate, Pyridine, Cisplatin, L-a-Phosphatidylcholine, Sephadex G-25and 1,2-Phenylenediamine were bought from Sigma-Aldrich.1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Amino(PolythyleneGlycol)2000] and the mini handheld Extruder kit (including 0.2 μmWhatman Nucleopore Track-Etch Membrane, Whatman filter supports and 1.0mL Hamiltonian syringes) were bought from Avanti Polar Lipids Inc.Anhydrous solvent DMF was supplied by Acros Organics. PhosphotungsticAcid was from Ted Pella, Inc. Analytical thin-layer chromatography (TLC)was performed using precoated silica gel Aluminium sheets 60 F254 boughtfrom EMD Laboratories. Spots on the TLC plates were visualized usingalkanine permanganate or 6% Ninhydrin solution in Acetone. 1H NMR (300MHz) spectra were obtained on a Varian Mercury 300 spectrophotometer.The chemical shifts are expressed in parts per million (ppm) usingsuitable deuterated NMR solvents with reference to TMS at 0 ppm. MTSreagent was supplied by Promega. The cell viability assay and releasekinetic data were plotted using GraphPad Prism software. Each sample wasdone in triplicate.

Synthesis of (11):

1044 μL (15 eq) of ethylene diamine (12) was dissolved in 5.0 mLanhydrous DCM followed by cooling down to 0-5° C. with ice. 500.0 mg(1.0 eq) of Cholesteryl Chloroformate was dissolved in 5.0 mL anhydrousDCM and was added to the reaction mixture drop-wise over a period 15minutes with vigorous stirring and was continued overnight until itcomes to rt. The reaction was worked up using water (50 mL×3) and DCM(50 mL), followed by saturated Brine water wash. The organic layer wasdried over anhydrous Sodium Sulphate, and evaporated with the help of arotary evaporator. Light yellow colored clear oily product (13) wasseparated with 99.1% yield. 1H-NMR (300 MHz) d (ppm)=5.37 (s, 1H), 5.06(S, 1H), 4.49 (bs, 1H), 3.22-3.20 (m, 2H), 2.82-2.81 (m, 2H), 2.34-2.26(m, 2H), 2.02-1.83 (m, 6H), 1.54-0.84 (m, 37H) Synthesis of 15:

350 mg (0.74 mmol, 1 eq) of starting material (13) was dissolved in 5.0mL anhydrous DCM. To it 370.0 mg (3.7 mmols, 5 eq) of Succinic Anhydride(14) and catalytic amount of Pyridine was added. The stirring wascontinued for Id followed by work up in 0.1(N) HCl and DCM for severaltimes. The organic layer was dried over Sodium Sulphate and evaporatedto get white amorphous solid compound (15). Yield: 95% 1H-NMR (300 MHz)d (ppm)=7.72-7.70 (m, 1H), 7.54-7.53 (m, 1H), 5.37 (s, 1H), 5.07 (s,1H), 4.49 (bs, 1H), 4.22-4.19 (m, 2H), 3.36-3.30 (m, 4H), 2.68-2.33 (m,4H), 2.02-1.83 (m, 6H), 1.54-0.84 (m, 37H).

Synthesis of 16:

50 mg (0.166 mmol, 1 eq) of Cisplatin (16) was partially dissolved in10.0 mL of H2O. To it 28.0 mg (0.166 mmol, 1 eq) Silver Nitrate wasadded and the resulting reaction mixture was stirred at rt for Id. Itlooked milky white and Silver Chloride was removed by centrifuging at25000×g for 1 h. Synthesis of 7: 200 mg (0.35 mmol, 1.0 eq) of 5 wasdissolved in 5.0 mL DMF. To it 20.0 mL of product 6 (conc 5.0 mg/mL, 1.0eq) was added and stirred for Id. The reaction mixture was dried withthe help of a lyophilizer. The dried product (17) was used forlipo-nanoparticle synthesis without any further purification.

General Procedure of Synthesizing Lipo-Nano Articles:

10.0 mg of L-a-Phosphatidylcholine, 5.0 mg of Cholesterol (orPt(II)-cholesterol conjugate) and 1.0 mg of1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Amino(PolythyleneGlycol)2000] were dissolved in 10.0 mL DCM. It was evaporated into athin and uniform film with the help of a rotary evaporator. Afterthorough drying with pump it was hydrated with 1.0 mL H2O for 2 h at 60°C. The hydrated lipo-nanoparticles looked light yellow to white withlittle viscous texture. It was passed though Sephadex G-25 column andextruded at 65° C.

General Method of Pt(II) Quantification in Lipo-Nanoparticles

A measured amount of the extruded lipo-nanoparticle was heated at 100°C. in 1.2 mg/mL concentration of 1,2-Phenylenediamine in DMF for 2 h.Pt(II) amount was calculated by UV Spectrophotometry (Shimadzu 2450).

Release Kinetics

Concentrated drug loaded lipo-nanoparticles were suspended with buffer(or cell lysate) and sealed in a dialysis membrane (MW cutoff 1000,Spectrum Lab). The dialysis bags were incubated in 1.0 mL PBS buffer atroom temperature with gentle shaking. A 10 μL portion of the aliquot wascollected from the incubation medium at predetermined time intervals,and the released drug was quantified by UV Spectrophotometer (Shimadzu2450). The results are plotted as percentage release.

Sample Preparation for TEM

High resolution TEM images were obtained on a Jeol 2011 high contrastdigital TEM. For sample preparation, lacy carbon 300 mesh copper grids(Electron microscopy Science) were dipped in the aqueous solution of thelipo-nanoparticle. It was allowed to air dry followed by staining itwith 2% aqueous solution of Phosphotungstic acid. The size distributionof lipo-nanoparticles was studied by dynamic light scattering (DLS),which was performed at 26° C. on a Malvern Zetasizer DLS-system equippedwith a He—Ne laser.

Cell Viability Assay

In a 96 well plate, 2×10³ cells were plated. After 4 h, cells weretreated with different concentrations of free drug orlipo-nanoparticles. Cells without any treatment were kept as control.After 48 h, cell viability was assessed using standard MTS assayaccording to manufacturer's instructions.

In Vivo Efficacy and Toxicity Studies

BALB/c mice were inoculated s.c. with 1×10⁵ of 4T1 breast tumor cells in100 μL PBS on right flank of mice. Treatment with different anticanceragents either free or entrapped in nanoparticles was started on day whentumor volume reached 200 mm³. Typically the animals received free drugalone or in nanoparticles through i.v route every alternate day fortotal of three dosages. Once the tumor volume reached 2000 mm3 incontrol group, mice were sacrificed. Tumor, kidney, spleen, lung, liverwere harvested and processed for paraffin embedding and sectioning.

All patents and publications cited herein are hereby incorporated byreference.

What is claimed:
 1. A biocompatible conjugated polymer nanoparticlecomprising: a copolymer backbone; a plurality of sidechains covalentlylinked to said backbone; and a plurality of platinum compoundsdissociably linked to said backbone.
 2. The nanoparticle of claim 1,wherein said plurality of platinum compounds is selected from Pt(II)compounds, Pt(IV) compounds, and any combinations thereof.
 3. Thenanoparticle of claim 1, wherein said copolymer comprises maleic acidmonomers.
 4. The nanoparticle of claim 1, wherein said sidechains areselected from the group consisting of polymers, monosaccharides,dicarboxylic acids, polyethylene glycol (PEG), and combinations thereof.5. The nanoparticle of claim 1, wherein said platinum compound is aPt(II) compound selected from the group consisting of cisplatin,oxaliplatin, carboplatin, paraplatin, sartraplatin, and combinationsthereof.
 6. The nanoparticle of claim 1, wherein at least one of saidplurality of platinum compounds is linked to the backbone by at leastone coordination bond, wherein the coordination bond is between anoxygen of the backbone and the platinum atom of the platinum compound.7. The biocompatible conjugated polymer nanoparticle of claim 1, whereinsaid copolymer is poly(isobutylene-alt-maleic acid) comprising 25 to 50monomer units; wherein said plurality of sidechains covalently linked tosaid backbone are PEG having a molecular weight of from 1000 to 3000Dalton and wherein the number of said PEG sidechains corresponds tobetween 50% and 100% of the number of monomeric units of said polymerbackbone; and wherein the number of said cisplatin sidegroups is between25% and 75% of the number of monomeric units of said polymer backbone.8. The biocompatible conjugated polymer nanoparticle of claim 1, whereinsaid copolymer is poly(isobutylene-alt-maleic acid) comprising from 25to 50 monomers; wherein said plurality of sidechains covalently linkedto said backbone are glucosamine; and wherein the number of saidglucosamine sidechains is between 50% and 100% of monomeric units ofsaid polymer backbone; and wherein the number of said cisplatinsidegroups is between 25% and 75% of the number of monomeric units ofsaid polymer backbone.
 9. A carboxylic acid-platinum compound complexconjugated nanoparticle comprising: a carboxylic acid-platinum compoundcomplex; and a plurality of lipid-polymer chains, wherein the carboxylicacid portion of said carboxylic acid-platinum compound complex iscovalently bound to said lipid-polymer chains.
 10. The nanoparticle ofclaim 9, wherein the carboxylic acid is maleic acid.
 11. Thenanoparticle of claim 9, wherein the polymer is PEG.
 12. Thenanoparticle of claim 9, wherein the platinum compound is a Pt(II)compound selected from the group consisting of cisplatin, oxaliplatin,carboplatin, paraplatin, sartraplatin, and combinations thereof.
 13. Thenanoparticle of claim 9, wherein the platinum compound loading is from1%-30%.
 14. A dicarbonyl-lipid compound having the structure


15. A vesicle, micelle, liposome or nanoparticle compound comprising adicarbonyl-lipid compound of claim 14 and a platinum compound, whereinthe platinum compound is dissociably linked to the compound of claim 14.16. The nanoparticle of claim 15, wherein the platinum compound isselected from Pt(II) compounds, Pt(IV) compounds, and any combinationsthereof.
 17. The nanoparticle of claim 16, wherein said platinumcompound is a Pt(II) compound selected from the group consisting ofcisplatin, oxaliplatin, carboplatin, paraplatin, sartraplatin, andcombinations thereof.
 18. A nanoparticle compound comprising abiocompatible polymer, wherein the polymer comprises at least onemonomer having the formula —CH(CO₂H)—R—CH(C(O)R′)—, wherein R is a bondor a C₁-C₆ alkylene, where the alkylene can comprise one or more doubleor triple bonds; and R′ is a substituted nitrogen atom.
 19. Thenanoparticle of claim 18, wherein R′ is

or —NH(CH₂CH₂O)_(m)CH₃, wherein m is 1-150.
 20. The nanoparticle ofclaim 18, further comprising a bioactive agent.
 21. A biocompatiblepolymer comprising: a copolymer backbone; a plurality of sidechainscovalently linked to the backbone; and a plurality of platinum compoundsdissociably linked to the backbone, wherein at least one of saidplurality of platinum compounds is linked to said backbone through atleast one coordination bond and said coordination bond is between anoxygen of the backbone and the platinum atom of the platinum compound.22. The biocompatible polymer of claim 21, wherein said oxygen is acarbonyl oxygen or an amide oxygen.
 23. The biocompatible polymer ofclaim 21, wherein said plurality of platinum compounds is selected fromPt(II) compounds, Pt(IV) compounds, and any combinations thereof. 24.The biocompatible polymer of claim 23, wherein said platinum compound isa Pt(II) compound selected from the group consisting of cisplatin,oxaliplatin, carboplatin, paraplatin, sartraplatin, and combinationsthereof.
 25. The biocompatible polymer of claim 21, wherein thecopolymer backbone comprises from 2 to 100 monomer units.
 26. Thebiocompatible polymer of claim 21, wherein the copolymer backbone ispoly(isobutylene-alt-maleic acid) (PIMA).
 27. The biocompatible polymerof claim 21, wherein the copolymer backbone comprises at least onemonomer having the formula —CH(CO₂H)—R—CH(C(O)R′)—,—CH(CO₂H)—R—CH(C(O)R′)CH₂C(Me₂)-, or —CH(C(O)R′)—R—CH(CO₂H)—CH₂C(Me₂)-wherein R is a bond, C₁-C₆ alkylene, where the alkylene can comprise oneor more double or triple bonds; and R′ is a substituted nitrogen atom.28. The biocompatible polymer of claim 21, wherein said sidechains areselected from the group consisting of polymers, monosaccharides,dicarboxylic acids, and combinations thereof.
 29. The biocompatiblepolymer of claim 21, wherein the number of sidechains correspondsbetween 50% and 100% of the number of monomeric units of said polymerbackbone.
 30. The biocompatible polymer of claim 21, wherein the numberof said platinum compounds corresponds between 10% and 100% of thenumber of monomeric units of said polymer backbone.
 31. Thebiocompatible polymer of claim 21, wherein the biocompatible polymercomprises: a poly(isobutylene-alt-maleic acid) backbone, wherein saidbackbone contains 25 to 50 monomer units; a plurality of PEG sidechainsor glucosamine sidechains covalently linked to said backbone, whereinsaid PEG sidechains have a molecular weight of from 1000 to 3000 Daltonand wherein the number of said PEG sidechains or glucosamine side chainscorresponds to between 50% and 100% of the number of monomeric units ofsaid polymer backbone; and a plurality of cisplatin sidegroupsdissociably linked to said backbone wherein the number of said cisplatinsidegroups is between 25% and 75% of the number of monomeric units ofsaid polymer backbone.
 32. The biocompatible polymer of claim 21,wherein the biocompatible polymer is


33. A method of treating cancer or metastasis comprising: administeringto a subject in need thereof an effective amount of a nanoparticle ofclaim 1.